Hyaluronate synthase gene and uses thereof

ABSTRACT

Disclosed are DNA sequences encoding hyaluronic acid synthase that are employed to construct recombinant cells useful in the production of hyaluronata synthase and hyaluronic acid (HA). In preferred aspects, chromosomal DNA encoding the HA synthase gene, hasA, was cloned from a  Streptococcus pyoganas  genomic library. These vectors were used to transform host cells such as  E. coli  and acapsular Streptococci to produce hyaluronic acid. Resultant transformants were screened to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies were selected and employed in the production of hyaluronic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/499,316, filedAug. 4, 2006; which is a continuation of U.S. Ser. No. 11/321,991, filedDec. 29, 2005, now abandoned; which is a continuation of U.S. Ser. No.11/024,426, filed Dec. 30, 2004, now U.S. Pat. No. 7,026,159, issuedApr. 11, 2006; which is a continuation of U.S. Ser. No. 10/124,222,filed Apr. 15, 2002, now U.S. Pat. No. 6,855,502, issued Feb. 15, 2005;which is a continuation of U.S. Ser. No. 09/146,893, filed Sep. 3, 1998,now U.S. Pat. No. 6,455,304, issued Sep. 24, 2002; which is acontinuation of U.S. patent application Ser. No. 08/270,581, filed Jul.1, 1994, now abandoned; the contents of each of which are herebyexpressly incorporated herein by reference.

The government owns certain rights in the present invention pursuant togrant number GM35978 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a nucleic acid encoding the enzymehyaluronate synthase, and to the use of this nucleic acid in thepreparation of recombinant cells for the production of the hyaluronatesynthase enzyme and hyaluronic acid. Hyaluronate is also known ashyaluronic acid or hyaluronan.

2. Description of the Related Art

The incidence of streptococcal infections is a major health and economicproblem worldwide, particularly in developing countries (Rotta, 1988).One reason for this is due to the ability of Streptococcal bacteria togrow undetected by the bodys phagocytic cells (i.e., macrophages andpolymorphonuclear cells (PMNs). These cells are responsible forrecognizing and engulfing foreign microorganisms. One effective way thebacteria evade surveillance is by coating themselves with polysaccharidecapsules, such as hyaluronic acid (HA) capsules. (Kendall et al., 1937).Since HA is generally nonimmunogenic (Quinn and Singh, 1957), theencapsulated bacteria do not elicit an immune response and are,therefore, not targeted for destruction. Moreover, the capsule exerts anantiphagocytic effect on PMNs in vitro (Hirsch, et al., 1960) andprevents attachment of Streptococcus to macrophages (Whitnack, et al.,1981). Precisely because of this, in group A and group C Streptococci,the HA capsules are major virulence factors in natural and experimentalinfections (Kass, et al., 1944; Wessels, et al., 1991). Group AStreptococcus are responsible for numerous human diseases includingpharyngitis, impetigo, deep tissue infections, rheumatic fever and atoxic shock-like syndrome (Schaechter, et al., 1989). The group CStreptococcus equisimilis is responsible for osteomyelitis (Earson,1986), pharyngitis (Benjamin, et al., 1976), brain abscesses (Dinn,1971), and pneumonia (Rizkallah, et al., 1988; Siefkin, et al., 1983).

Structurally, HA is a high molecular weight linear polysaccharide ofrepeating disaccharide units consisting of N-acetylglucosamine (GlcNAc)and glucuronic acid (GlcA) (Laurent and Fraser, 1992). HA is the onlyglycosaminoglycan synthesized by both mammalian and bacterial cellsparticularly groups A and C Streptococci. Some Streptococcus strainsmake HA which is secreted into the medium as well as HA capsules. Themechanism by which these bacteria synthesize HA is of interest since theproduction of the HA capsule is a very efficient and clever way thatStreptococci use to evade surveillance by the immune system.

HA is synthesized by both mammalian and Streptococcus cells by theenzyme hyaluronate synthase, that has been localized to the plasmamembrane of Streptococcus (Markovitz, et al., 1962). The synthesis of HAin these organisms is a multi-step process. Initiation involves bindingof an initial precursor, UDP-GlcNAc or UDP-GlcA. This is followed byelongation which involves alternate addition of the two sugars to thegrowing oligosaccharide chain. The growing polymer is extruded acrossthe bacterial plasma membrane region of the cell wall and into theextracellular space. Although the HA biosynthetic system was one of thefirst membrane heteropolysaccharide synthetic pathways studied, themechanism of HA synthesis is still not understood. This may be becausein vitro systems developed to date are inadequate in that de novobiosynthesis of HA has not been accomplished. Chain elongation but notnew chain initiation occurs.

The direction of HA polymer growth is a matter of disagreement. Additionof the monosaccharides could be to the reducing (Prehm, 1983) ornonreducing (Stoolrniller, et al., 1969) end of the growing HA chain. Inaddition, other questions that need to be addressed are (i) whethernascent chains are linked covalently to a protein, to UDP or to a lipidintermediate, (ii) whether chains are initiated using a primer, and(iii) the mechanism by which the mature polymer is extruded through theplasma membrane of the Streptococcus. Understanding the mechanism of HAbiosynthesis may allow development of alternative strategies to controlStreptococcal infections by interfering in the process.

Group C S. equisimilis strain D181 synthesizes, and secretes HA.Investigators have used this strain and group A strains, such as A111,to study the biosynthesis of HA and to characterize the HA-synthesizingactivity in terms of its divalent cation requirement (Stoolmiller, etal., 1969), precursor (UDPC-GlcNAc and UDP-G1cUA) utilization (Ishimoto,et al., 1967; Markovitz, et al., 1959) and optimum pH (Stoolmillér, etal., 1969). The HA synthase enzyme has been studied for approximately 30years, but has not yet been identified or purified. Although a 52-kDprotein has been putatively suggested as the HA synthase (Prehm, et al.,1986), this report is now known to be in error. Furthermore, no one hassuccessfully purified to homogeneity an active enzyme. Moreover, it'snot clear whether a bona fide HA synthase molecule is all that is neededfor the generation of hyaluronic acid, or whether it might act inconcert with other cellular components or subunits. Thus, totally exvivo methods of producing HA have not been forthcoming.

Typically, HA has been prepared commercially by isolation from eitherrooster combs or extracellular media from Streptococcal cultures. Onemethod which has been developed for preparing HA is through the use ofcultures of HA-producing streptococcal bacteria. U.S. Pat. No.4,517,295, describes such a procedure, wherein HA-producing Streptococciare fermented under anaerobic conditions in a CO₂-enriched growthmedium. Under these conditions, HA is produced and can be extracted fromthe broth. It is generally felt that isolation of HA from rooster combis laborious and difficult, since one starts with HA in a less purestate. The advantage of isolation from rooster comb is that the HAproduced is of higher molecular weight. However, preparation of HA bybacterial fermentation is easier, since the HA is of higher purity tostart with. Usually, however, the molecular weight of HA produced inthis way is smaller than that from rooster combs. Therefore, a techniquethat would allow the production of high molecular weight HA by bacterialfermentation would be an improvement over existing procedures.

High molecular weight HA has a wide variety of usefulapplications—ranging from cosmetics to eye surgery (Laurent and Fraser,1992). Due to its potential for high viscosity and its highbiocompatibility, HA finds particular application in eye surgery as areplacement for vitreous fluid. HA has also been used to treatracehorses for traumatic arthritis by intra-articular injections of HA,in shaving cream as a lubricant, and in a variety of cosmetic productsdue to its physicochemical properties of high viscosity and its abilityto retain moisture for long periods of time. In general, the highermolecular weight the HA that is employed the better. This is because HAsolution viscosity increases with the average molecular weight of theindividual HA polymer molecules in the solution. Unfortunately, veryhigh molecular weight HA, such as that ranging up to 10⁷, has beendifficult to obtain by currently available isolation procedures.

To address these or other difficulties, there is a need for new methodsand constructs that can be used to produce HA having one or moreimproved properties such as greater purity or ease of preparation. Inparticular, there is a need to develop methodology for the production oflarger amounts of relatively higher molecular weight and purity HA thanis available from current technology. The present invention addressesone or more shortcomings in the art through the application ofrecombinant DNA technology.

SUMMARY OF THE INVENTION

The present invention involves the application of recombinant DNAtechnology to solving one or more problems in the art of hyaluronic acidpreparation. These problems are addressed through the isolation and useof a DNA segment encoding all or a portion of the hyaluronate synthasegene, the gene responsible for HA chain biosynthesis. The gene wascloned from DNA of an appropriate microbial source and engineered intouseful recombinant constructs for the preparation of HA and for thepreparation of large quantities of the HA synthase enzyme itself.

The present invention, in a general and overall sense, concerns theisolation and characterization of a hyaluronate or hyaluronic acidsynthase gene, as may be used for the polymerization of gluouronic acidand N-acetylglucosamine into the glycosaminoglycan hyaluronic acid. Thepresent inventors have identified the hasA locus and have determined thesequence encoding the Hyaluronic acid synthase (HA synthase) gene fromStreptococcus. The hasA gene product, HasA, has been expressed inhomologous and heterologous cells, can be used to isolate hyaluronicacid synthase, and can be used for the production of hyaluronic acid.The hasA gene also provides a new probe to assess the potential ofbacterial specimens to produce hyaluronic acid.

The present invention encompasses a novel gene, hasA. The expression ofthis gene correlates with virulence of Streptococcal strains, byproviding a means of escaping immune surveillance. The term, “hyaluronicacid synthase”, “hyaluronate synthase”, “hyaluronan synthase” and “HAsynthäse”, are used interchangeably to describe an enzyme thatpolymerizes a glycosaminoglycan polysaccharide chain composed ofalternating glucuronic acid (GleVa) and N-Cacetylglucosamine (GlcNac)sugars.

Through the application of techniques and knowledge set forth herein,those of skill in the art will be able to obtain nucleic acid segmentsencoding an HA synthase gene. Through isolation of the HA gene, fromwhatever source is chosen, one will have the ability to realizesignificant advantages such as an ability to manipulate the host that ischosen to express the HA synthase gene, the fermentation environmentchosen for HA production, as well as genetic manipulation of theunderlying gene. As those of skill in the art will recognize, in lightof the present disclosure, this will provide additional significantadvantages both in the ability to control the expression of the gene andin the nature of the gene product that is realized.

Accordingly, the invention is directed to the isolation of DNA thatcomprises the HA synthase gene, whether it be from prokaryotic oreukaryotic sources. This is possible because the enzyme, and indeed thegene, is one found in both eukaryotes and some prokaryotes. Typicalprokaryotic sources will include Group A or Group C Streptococcussources such as S. pyogenes, S. equisimilis, or S. zooepidemicus.Eukaryotes are also known to produce HA (Mg and Schwartz, 1989) and thushave HA synthase genes that may be employed in connection with theinvention. For example, it is known that HA is produced in rooster combsby mesodermal cells of the rooster. These cells can be employed toisolate starting mRMA for the production of cDNA libraries by well knowntechniques, which can subsequently be screened by novel screeningtechniques set forth herein. Other eukaryotic sources that can beemployed include synovial chondrocytes and fibroblasts, dermalfibroblasts, and even trabecular-meshwork cells of the eye.

HA synthase-encoding nucleic acid segments of the present invention aredefined as being isolated free of total chromosomal or genomic DMA suchthat they may be readily manipulated by recombinant DNA techniques.Accordingly, as used herein, the phrase “substantially purified DNAsegment” refers to a DNA segment isolated free of total chromosomal orgenomic DNA and retained in a state rendering it useful for the practiceof recombinant techniques, such as DMA in the form of a discreteisolated DNA fragment, or a vector (e.g., plasmid, phage or virus)incorporating such a fragment.

A preferred embodiment of the present invention is a purified nucleicacid segment encoding HA synthase, wherein the segment encodes a proteinhaving an amino acid sequence in accordance with SEQ ID NO:2, or that iscapable of hybridizing to the nucleotide sequence of SEQ ID NO:1 understandard hybridization conditions as described herein. The nucleotidesegment of the present invention is a purified nucleic acid segment,further defined as including a nucleotide-sequence as shown in FIG. 7,and in accordance with SEQ ID NO:1.

In a more preferred embodiment the purified nucleic acid segmentconsists essentially of the nucleotide sequence of SEQ ID NO:1. As usedherein, the term “nucleic acid segment” and “DNA segment” are usedinterchangeably and refer to a DNA molecule which has been isolated freeof total genomic DNA of a particular species. Therefore, a “purified”DNA or nucleic acid segment as used herein, refers to a DNA segmentwhich contains an hasA coding sequence yet is isolated away from, orpurified free from, total genomic DNA, for example, total Streptococcuspyogenes or, for example, mammalian host genomic DNA. Included withinthe term “DNA segment”, are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified hasA generefers to a DNA segment including hasA coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case hasA, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HA synthase gene from prokaryotes such as S. pyogenes or S.equisimilis. One such advantage is that, typically, eukaryotic enzymesmay require significant post-translational modifications that can onlybe achieved in a eukaryotic host. This will tend to limit theapplicability of any eukaryotic HA synthase gene that is obtained.Moreover, those of ordinary skill in the art will likely realizeadditional advantages in terms of time and ease of genetic manipulationwhere a prokaryotic enzyme gene is sought to be employed. Theseadditional advantages include: (a) the ease of isolation of aprokaryotic gene because of the relatively small size of the genome and,therefore, the reduced amount of screening of the corresponding genomiclibrary, and (b) the ease of manipulation because the overall size ofthe coding region of a prokaryotic gene is significantly smaller due tothe absence of introns. Furthermore, if the product of the HA synthasegene (i.e., the enzyme) requires posttranslational modifications, thesewould best be achieved in a similar prokaryotic cellular environment(host) from which the gene was derived.

Preferably, DNA sequences in accordance with the present invention willfurther include genetic control regions which allow the expression ofthe sequence in a selected recombinant host. Of course, the nature ofthe control region employed will generally vary depending on theparticular use (e.g., cloning host) envisioned. For example, instreptococcal hosts, the preferred control region is the homologouscontrol region associated with the structural gene in its natural state.The homologous control region, in fact, may be coisolated directly withthe isolation of the HA synthase structural gene itself through thepractice of certain preferred techniques disclosed herein.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode an hasAgene, that includes within its amino acid sequence an amino acidsequence in accordance with SEQ ID NO:2. Moreover, in other particularembodiments, the invention concerns isolated DMA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anhasA gene corresponding to Streptococcus pyogenes hasA. Naturally, wherethe DNA segment or vector encodes a full length HasA protein, or isintended for use in expressing the NasA protein, the most preferredsequences are those which are essentially as set forth in SEQ ID NO:2.

Nucleic acid segments having HA synthase activity may be isolated by themethods described hereinabove. The term “a sequence essentially as setforth in SEQ ID NO:2” means that the sequence substantially correspondsto a portion of SEQ ID NO:2 and has relatively few amino acids which arenot identical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:2. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as agene having a sequence essentially as set forth in SEQ ID NO:2, and thatis associated with the ability of Streptococcus to produce HA and ahyaluronic acid coat. Accordingly, sequences which have between about70% and about 800%; or more preferably, between about 81% and about 90%;or even more preferably, between about 91% and about 99%; of amino acidswhich are identical or functionally equivalent to the amino acids of SEQID NO:2 will be sequences which are “essentially as set forth in SEQ IDNO:2”.

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:2, further defined as a recombinant vector. As used herein the term,“recombinant vector”, refers to a vector that has been modified tocontain a nucleic acid segment that encodes an HasA protein, or fragmentthereof. The recombinant vector may be further defined as an expressionvector comprising a promoter operatively linked to said NasA encodingnucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising an hasA gene. Thepreferred recombinant host cell may be a prokaryotic cell. In anotherembodiment, the recombinant host cell is a eukaryotic cell. As usedherein, the term “engineered” or “recombinant” cell is intended to referto a cell into which a recombinant gene, such as a gene encoding hasA,has been introduced. Therefore, engineered cells are distinguishablefrom naturally occurring cells which do not contain a recombinantlyintroduced gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinantly introduced genes willeither be in the form of a cDNA gene, a copy of a genomic gene, or willinclude genes positioned adjacent to a promoter not naturally associatedwith the particular introduced gene.

Where one desires to use a host other than Streptococcus, as may be usedto produce recombinant HA synthase, it may be advantageous to employ aprokaryotic system such as E. coli, B. subtilis, Lactococcus sp., oreven eukaryotic systems such as yeast or Chinese hamster ovary, Africangreen monkey kidney cells, VERO cells, or the like. Of course, wherethis is undertaken, it will generally be desirable to bring the HAsynthase gene under the control of sequences which are functional in theselected alternative host. The appropriate DNA control sequences, aswell as their construction and use, are generally well known in the artas discussed in more detail herein below.

In preferred embodiments, the HA synthase-encoding DNA segments furtherinclude DNA sequences, known in the art functionally as origins ofreplication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which HA synthase DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin Streptococcus hosts. However, for more versatility of cloned DNAsegments, it may be desirable to alternatively or even additionallyemploy origins recognized by other host systems whose use iscontemplated (such as in a shuttle vector).

The isolation and use of other replication origins such as the SV40,polyoma or bovine papilloma virus origins, which may be employed forcloning in a number of higher organisms, are well known (Fiers, et al.,1978). In certain embodiments, the invention may thus be defined interms of a recombinant transformation vector which includes the HAsynthase gene sequence together with an appropriate replication originand under the control of selected control regions.

In accordance with the present invention, the HA synthase gene, whenfrom a prokaryotic source such as a Streptococcal source, is obtained bythe following general steps. First, the genetic loci are identified bytransposon insertional mutagenesis. One such transposon system is theTN916, obtainable from the transposon donor strain E. faecalis CG110,which was used to mutate the mucoid strain of Streptococcus pyogenesS43. Mutants were isolated and the genomic DNA surrounding thetransposon was sequenced and used to derive oligonucleotides for use incloning the wild-type gene. Phage libraries were screened, and twoclones, λ1X and λ2Y, were obtained that contained the predictedsequence. The locus was characterized by restriction mapping andsouthern blot analysis.

Thus, although the present invention is exemplified in terms of clonesscreened via transposon mediated mutagenesis, it will be appreciated bythose of skill in the art that other means may be used to obtain thehasA gene, in light of the present disclosure. For example, polymerasechain reaction produced DNA fragments may be obtained which contain fullcomplements of genes from a number of sources, including other strainsof Streptococcus or from eukaryotic sources, such as cDNA libraries.Virtually any molecular cloning approach may be employed for thegeneration of DNA fragments in accordance with the present invention.Thus, the only limitation generally on the particular method employedfor DNA isolation is that the isolated nucleic acids should encode abiologically functional equivalent HA synthase.

Once the DNA has been isolated it is ligated together with a selectedvector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepBluescript™, pSA3, lambda, SV40, polyoma, adenovirus, bovine papillomavirus and retroviruses. However, it is believed that particularadvantages will ultimately be realized where vectors capable ofreplication in both Lactococcus or Bacillus strains and E. coli areemployed.

Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti (Dao, et al., 1985) or the pATl9 vector of TrieuCuot, et al.(1991), allow one to perform clonal colony selection in an easilymanipulated host such as E. coli, followed by subsequent transfer backinto a Lactococcus or Bacillus strain for production of HA. This isadvantageous in that one can augment the Lactococcus or Bacillusstrain's ability to synthesize HA through gene dosaging (i.e., providingextra copies of the HA synthase gene by amplification) and/or inclusionof additional genes to increase the availability of HA precursors. Theinherent ability of Streptococci to synthesize HA can also be augmentedthrough the formation of extra copies, or amplification, of the plasmidthat carries the HA synthase gene. This amplification can account for upto a 10-fold increase in plasmid copy number and, therefore, the HAsynthase gene copy number.

Another procedure that would further augment HA synthase gene copynumber is the insertion of multiple copies of the gene into the plasmid.This extra amplification would be especially feasible, since thebacterial HA synthase gene size is small. In any event, the chromosomalDNA-ligated vector is employed to transfect the host that is selectedfor clonal screening purposes such as E. coli, through the use of avector that is capable of expressing the inserted DNA in the chosenhost.

Where a eukaryotic source such as dermal or synovial fibroblasts orrooster comb cells is employed, one will desire to proceed initially bypreparing a cDNA library. This is carried out first by isolation of mRNAfrom the above cells, followed by preparation of double stranded cDNAusing an enzyme with reverse transcriptase activity and ligaton with theselected vector. Numerous possibilities are available and known in theart for the preparation of the double stranded cDNA, and all suchtechniques are believed to be applicable. A preferred technique involvesreverse transcription. Once a population of double stranded cDNAs isobtained, a cDNA library is prepared in the selected host by acceptedtechniques, such as by ligation into the appropriate vector andamplification in the appropriate host.

Due to the high number of clones that are obtained, and the relativeease of screening large numbers of clones by the techniques set forthherein, one may desire to employ phage expression vectors, such as λgtllor λgtl2, for the cloning and expression screening of cDNA clones.

Due to the general absence of correct information regarding the HAsynthase enzyme, traditional approaches to clonal screening, such asoligonucleotide hybridization or immunological screening, was notavailable. Accordingly, it was necessary for the inventors to use analternative approach based on phenotype to screen and select for the HAsynthase that rely on abrogating expression of HA synthase activity. Themethods which were developed can be applied to screen the selected host,regardless of whether a eukaryotic or prokaryotic gene is sought. Onemethod involves the application of a dye exclusion technique to identifyclones which contain HA. The typical dye employed, India ink, isexcluded from an HA capsule and allows visualization of HA by negativestaining. A second method involves positive staining, such as withAlcian Blue, to identify HA producing clones. Alcian Blue binds to andstains polyanionic molecules such as HA (Scott, et al., 1964). However,in that India ink or Alcian Blue is not entirely specific for HA, thepresent inventors employed additional screening methods as described inExamples I and II.

A variety of additional screening and validation procedures are also setforth herein that can variously be employed to identify the presence ofeither the HA enzyme or its HA product as a means for identifyingpositive clones or negative clones (mutants). These procedures includedthe use of Percoll gradient centrifugation and the ability of membranefractions from candidate clones to incorporate authentic radiolabeledsugar nucleotides into high molecular weight HA.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1”, is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table I, and also refers tocodons that encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression and enzyme activityis concerned. The addition of terminal sequences particularly applies tonucleic acid sequences which may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region or may include various internal sequences, which are knownto occur within genes.

Allowing for the degeneracy of the genetic code, sequences which havebetween about 70% and about 80%; or more preferably, between about 80%and about 90%; or even more preferably, between about 90% and about 99%;of nucleotides which are identical to the nucleotides of SEQ ID NO:1will be sequences which are “essentially as set forth in SEQ ID NO:1”.Sequences which are essentially the same as those set forth in SEQ IDNO:1 may also be functionally defined as sequences which are capable ofhybridizing to a nucleic acid segment containing the complement of SEQID NO:1 under relatively stringent conditions. Suitable relativelystringent hybridization conditions will be well known to those of skillin the art and are clearly set forth herein, for example conditions foruse with southern and northern blot analysis.

The term “standard hybridization conditions” as used herein, is used todescribe those conditions under which substantially complementarynucleic acid segments will form standard Watson-Crick base-pairing. Anumber of factor are known that determine the specificity of binding orhybridization, such as pH, salt concentration, the presence ofchaotropic agents (e.g., formamide and dimethyl sulfoxide), the lengthof the segments that are hybridizing, and the like.

For use with the present invention, standard hybridization conditionsfor relatively large segments, that is segments longer than about 100nucleotides, will include a hybridization mixture having between about0.3 to 0.6 M NaCl, a divalent cation chelator (e.g., EDTA at about 0.05mM to about 0.5 mM), and a buffering agent (e.g., Na2PO4 at about 10 mMto 100 mM, pH 7.2), at a temperature of about 65° C. The preferredconditions for hybridization are a hybridization mixture comprising 0.5M NaCl, 5 mM EDTA, 0.1 M Na₂PO₄, pH 7.2 and 1% N-lauryl sarcosine, at atemperature of 65° C. Naturally, conditions that affect thehybridization temperature, such as the addition of chaotropic agents,such as formamide, will be known to those of skill in the art, and areencompassed by the present invention.

When it is contemplated that shorter nucleic acid segments will be usedfor hybridization, for example fragments between about 14 and about 100nucleotides, salt and temperature conditions will be altered to increasethe specificity of nucleic acid segment binding. Preferred conditionsfor the hybridization of short nucleic acid segments include loweringthe hybridization temperature to about 37° C., and increasing the saltconcentration to about 0.5 to 1.5 M NaCl with 1.5 M NaCl beingparticularly preferred.

TABLE I CODON DEGENERACY Amino Acids Codons Alanine Ala A GCA GCC GCGGCU Cysteine Gys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid GluE GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CDU Methionine Met M AUGAsparagine Aen N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1. Nucleic acid sequences which are “complementary” arethose which are capable of base-pairing according to the standardWatson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 under relativelystringent conditions such as those described herein in Example III.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared whichinclude a short stretch complementary to SEQ ID NO:1, such as about 10to 15 or 20, 30, or 40 or so nucleotides, and, which are up to 10,000 or5,000 base pairs in length, with segments of 3,000 being preferred incertain cases. DNA segments with total lengths of about 1,000, 500, 200,100 and about 50 base pairs in length are also contemplated to beuseful.

A preferred embodiment of the present invention is a nucleic acidsegment which comprises at least a 10-14 nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1. In a more preferred embodiment the nucleic acid is furtherdefined as comprising at least a 20 nucleotide long stretch, a 30nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide longstretch, a 200 nucleotide long stretch, a 500 nucleotide long stretch, a1000 nucleotide long stretch, a 1500 nucleotide long stretch, or atleast a 1441 nucleotide long stretch which corresponds to, or iscomplementary to, the nucleic acid sequence of SEQ ID NO:1. The nucleicacid segment may be further defined as having the nucleic acid sequenceof SEQ ID NO:1.

A related embodiment of the present invention is a nucleic acid segmentwhich comprises at least a 10-14 nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1, further defined as comprising a nucleic acid “fragment of ˜ipto 10,000 basepairs in length”. A more preferred embodiment if a nucleicacid fragment comprising from 14 nucleotides of “SEQ ID NO:1 up to 5,000basepairs in length, 3,000 basepairs in length, 1,000 basepairs inlength, 500 basepairs in length, or 100 basepairs in length”.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NOS:1and 2. Recombinant vectors and isolated DNA segments may thereforevariously include the hasA coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides which neverthelessinclude hasA coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent HasA proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HasAprotein or to test HasA mutants in order to examine HA synthase activityat the molecular level.

A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to an HasA protein composition, wherein the HasAprotein is purified to any degree relative to its naturally-obtainablestate, i.e., in this case, relative to its purity within a prokaryoticcell extract. A preferred cell for the isolation of HasA protein is aStreptococcus pyoganes cell, however, HasA protein may also be isolatedfrom other members of the Streptococcus genus, patient specimens,recombinant cells, infected tissues, isolated subpopulation of tissuesthat contain high levels of hyaluronate in the extracellular matrix, andthe like, as will be known to those of skill in the art, in light of thepresent disclosure. A purified HasA protein composition therefore alsorefers to a polypeptide having the amino acid sequence of SEQ ID NO:2,free from the environment in which it may naturally occur.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the HasA protein coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

Turning to the expression of the hasA gene whether from genomic DNA, ora cDNA one may proceed to prepare an expression system for therecombinant preparation of HasA protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression. For example, one may prepare a HasA-GST(glutathione-S-transferase) fusion protein that is a convenient means ofbacterial expression. However, it is believed that virtually anyexpression system may be employed in the expression of HasA.

HasA may be successfully expressed in eukaryotic expression systems,however, the inventors aver that bacterial expression systems can beused for the preparation of HasA for all purposes. The cDNA for HasA maybe separately expressed in bacterial systems, with the encoded proteinsbeing expressed as fusions with B-galactosidase, avidin, ubiquitin,Schistosoma japonicum glutathione S-transferase, maltose-bindingprotein, polyhistidine-tags, epitope-tags (e.g., myc and FLAG) and thelike. It is believed that bacterial expression will ultimately haveadvantages over eukaryotic expression in terms of ease of use andquantity of materials obtained thereby.

It is proposed that transformation of host cells with DNA segmentsencoding HasA will provide a convenient means for obtaining an HasAprotein. It is also proposed that cDNA, genomic sequences, andcombinations thereof, are suitable for eukaryotic expression, as thehost cell will, of course, process the genomic transcripts to yieldfunctional mRNA for translation into protein.

Another embodiment of the present invention is a method of preparing aprotein composition comprising growing recombinant host cell comprisinga vector that encodes a protein which includes an amino acid sequence inaccordance with SEQ ID NO:2. The host cell will be grown underconditions permitting nucleic acid expression and protein productionfollowed by recovery of the protein so produced. The production of HAsynthase and HA, including: the host cell, conditions permitting nucleicacid expression, protein production and recovery will be known to thoseof skill in the art in light of the present disclosure of the hasA gene,and the hasA gene protein product HasA, and by the methods described inExamples III, IV, and V.

Preferred hosts for the expression of hyaluronic acid are prokaryotes,such as S. pyogenes, S. equisimilis, and other suitable members of theStreptococcus species. However, it is also contemplated that HA may besynthesized by heterologous host cells expressing HA synthase, such asspecies members of the Bacillus, Salmonella, Pseudomonas, Enterococcus,or even Escherichia genus. A most preferred host for expression of theHA synthase of the present invention is a bacteria transformed with thehasA gene of the present invention, such as Lactococcus, Bacillussubtilis or S. pyogenes.

It is similarly believed that almost any eukaryotic expression systemmay be utilized for the expression of hasA e.g., baculovirus-based,glutamine synthase-based, dihydrofolate reductase-based systems, SV-40based, adenovirus-based, cytomegalovirus-based, and the like, could beemployed. For expression in this manner, one would position the codingsequences adjacent to and under the control of the promoter. It isunderstood in the art that to bring a coding sequence under the controlof such a promoter, one positions the 5′ end of the transcriptioninitiation site of the transcriptional reading frame of the proteinbetween about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of)the chosen promoter.

Where eukaryotic expression is contemplated, one will also typicallydesire to incorporate into the transcriptional unit which includes thehasA gene, an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) ifone was not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

It is contemplated that virtually any of the commonly employed hostcells can be used in connection with the expression of hasA inaccordance herewith. Examples of preferred cell lines for expressing theHA synthase gene of the present invention include cell lines typicallyemployed for eukaryotic expression such as 239, AtT-20, HepG2, VERO,HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.

This will generally include the steps of providing a recombinant hostbearing the recombinant DNA segment encoding the HA synthase enzyme andcapable of expressing the enzyme; culturing the recombinant host inmedia under conditions that will allow for transcription of the clonedHA gene and appropriate for the production of the hyaluronic acid; andseparating and purifying the HA synthase enzyme or the secretedhyaluronic acid from the recombinant host.

Generally, the conditions appropriate for expression of the cloned HAsynthase gene will depend upon the promoter, the vector, and the hostsystem that is employed. For example, where one employs the lacpromoter, one will desire to induce transcription through the inclusionof a material that will stimulate lac transcription, such as IPTG. Whereother promoters are employed, different materials may be needed toinduce or otherwise up-regulate transcription. In addition, to obtainingexpression of the synthase, one will preferably desire to provide anenvironment that is conducive to HA synthesis by including appropriategenes encoding enzymes needed for the biosynthesis of sugar nucleotideprecursors, and by using growth media containing substrates for theprecursor-supplying enzymes, such N-acetylglucosamine (GlcMAc) andglucose (Glc).

One may further desire to incorporate the gene in a host which isdefective in the enzyme hyaluronidase, so that the product synthesizedby the enzyme will not be degraded in the medium. Furthermore, a hostwould be chosen to optimize production of HA. For example, a suitablehost would be one that produced large quantities of the sugar nucleotideprecursors to support the HA synthase enzyme and allow it to producelarge quantities of HA. Such a host may be found naturally or may bemade by a variety of techniques including mutagenesis or recombinant DNAtechnology. The genes for the sugar nucleotide synthesizing enzymes,particularly the UDP-Glc dehydrogenase required to produce UDP-GlcA,could also be isolated and incorporated in a vector along with the HAsynthase gene. A preferred embodiment of the present invention is a hostcontaining these ancillary recombinant genes and the amplification ofthese gene products thereby allowing for increased production of HA.

In the case where production of HA synthase is desired, the enzyme ispreferably synthesized in bacteria using the T7 expression system(Studier, et al., 1990). pT5 plasmids containing the HA synthase geneinserted adjacent to the philO promoter are transformed into E. colistain BL21(DE3)pLysS. In this strain the T7 gene encoding thebacteriophage RNA polymerase is under control of the E. coli lacZpromoter. Therefore, the polymerase can be induced by IPTG andtranscription of the HA synthase gene is, in turn, induced from the φ10promoter of the pT5 vector.

The means employed for culturing of the host cell is not believed to beparticularly crucial. For useful details, one may wish to refer to thedisclosure of U.S. Pat. Nos. 4,517,295; 4,801,539; 4,784,990; or4,780,414; all incorporated, herein by reference. Where a prokaryotichost is employed, such as S. pyoganes or S. equisimilis, one may desireto employ a fermentation of the bacteria under anaerobic conditions inCO₂-enriched broth growth media. This allows for a greater production ofHA than under aerobic conditions. Another consideration is thatStreptococcal cells grown anaerobically do not produce pyrogenicexotoxins. Appropriate growth conditions can be customized for otherprokaryotic hosts, as will be known to those of skill in the art, inlight of the present disclosure.

Once the appropriate host has been constructed, and cultured underconditions appropriate for the production of HA, one will desire toseparate the HA so produced. Typically, the HA will be secreted orotherwise shed by the recombinant organism into the surrounding media,allowing the ready isolation of HA from the media by known techniques.For example, HA can be separated from the media by filtering and/or incombination with precipitation by alcohols such as ethanol. Otherprecipitation agents include organic solvents such as acetone orquaternary organic ammonium salts such as cetyl pyridinium chloride(CPC).

A preferred technique for isolation of HA is described in U.S. Pat. No.4,517,295 in which the organic carboxylic acid, trichloroacetic acid, isadded to the bacterial suspension at the end of the fermentation. Thetrichloroacetic acid causes the bacterial cells to clump and die andfacilitates the ease of separating these cells and associated debrisfrom HA, the desired product. The clarified supernatant is concentratedand dialyzed to remove low molecular weight contaminants including theorganic acid. The aforementioned procedure utilizes Millipore™filtration through filter cassettes containing 0.22 μm pore sizefilters. Diafiltration is continued until the conductivity of thesolution decreases to approximately 0.5 mega-ohms.

The concentrated HA is precipitated by adding an excess of reagent gradeethanol or other organic solvent and the precipitated HA is then driedby washing with ethanol and vacuum dried, lyophilized or spray dried toremove alcohol. The HA can then be redissolved in a borate buffer, pH 8,and precipitated with CPC or certain other organic ammonium salts suchas CETAB, a mixed trimethyl ammonium bromide solution at 4 degree(s) C.The precipitated HA is recovered by coarse filtration, resuspended in 1M NaCl, diafiltered and concentrated as further described in the abovereferenced patent. The resultant HA is filter sterilized and ready to beconverted to an appropriate salt, dry powder or sterile solution,depending on the desired end use.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Restriction map of the streptococcal HA biosynthesis locus.EcoRI (E) restriction sites on the wild-type S43 chromosome areillustrated with respect to the relevant Tn insertion and the minimalextent of the associated deletion (hatched box). The region of DNAcapable of directing HA synthesis in mutants and heterologous species,pPD41 5 is shown. Two HindIII (H), two PstI (P) and one BglII (B) siteswere found. KpnI, BamHI, SalI, Sad, Smal, SphI, and XbaI did not cut thepPD41 5 insert. The multiple cloning site (M) is the result of fusion ofthe deleted, blunt ended DNA and the pAT19 M. The inserts of the initialclones pB3 and pPD41 are shown above the genomic map. The large EcORIfragment on the extreme left is ˜20 kb and not shown to scale.

FIG. 2: Visualization of HA capsules in transformed bacteria by lightmicroscopy. These photomicrographs of early log cultures stained withIndia ink (Collins and Lyne, 1976) were taken on a Leitz Laboruxmicroscope at 1000× magnification. The results depict the ability ofplasmids pPD41 or pPD41 5, but notpAT19 alone or pPD41 7, to direct HAcapsule biosynthesis after transformation into the acapsular S. pyoganasmutant S43Tn7 or into normally acapsular E. faecalis. The bright halosurrounding the cells is the HA capsule. Ovine testicular hyaluronidasetreatment destroyed the capsule. Panels: A, wild-type S43; B,S43Tn7(pATl9); C, S43Tn7(pPD41); D, E. faecalis(pPD41 5); E, E.faecalis(pPD41 7); F, E., faecalis (pPD41 5) treated with 500 units/mlhyaluronidase (type V, 2000 u/mg) for 30 min at 37° C.

FIG. 3: Page analysis of polysaccharides produced by various transformedbacteria. Authentic HA (std) and polysaccharides purified from cellcultures as described by DeAngelis, et al. (1993a) were electrophoresedon a 4% gel and stained with Alcian Blue. Strains without pPD41 or pPD415 do not produce HA. The majority of the polymer population in eachsample migrated similarly to high MW HA (lanes F,G). The bracket on theright marks the extent of staining of the low MW HA standard, which didnot photograph well (lane H). The arrowhead indicates the top of thegel. Streptomyces hyaluronate lyase [HAase] treatment (20 units, 15 min)completely degraded the bacterial products. One μg (by carbazole assay)samples were loaded in lanes A-H and 8 μg samples were loaded in I-K.Lanes: A, S43; B, S43Tn7(pATl9); C, S43Tn7(pPD41); D, E. faecalis(pPD417); E, E. faecalis(pPD41 5); F, native HA, viscosity=13,172; G, HA withviscosity=1,589; H, HA with viscosity=20; I, HAase-treated sample C; J,HAasetreated sample E; K, HAase-treated sample F.

FIG. 4: SDS-Page analysis of proteins synthesized by pPD41 deletionplasmids in E. coli minicells. Minicells labeled with [³⁵S]Met/Cys werelysed by boiling in SDS-sample buffer and electrophoresed on a 10% gel.Cells containing the pPD41 5 plasmid produce HA and two proteins areseen on this autoradiogram (24 hr exposure) at 42 and 45 kDa (lane 1,positions marked with arrows) that are not produced by vector alone(lane 3). Cells containing the pPD41 7 plasmid do not produce HA andonly synthesize the 45 kDa protein (lane 2) Standards (BioRad, low MW)are shown in kDa.

FIG. 5: Tn Mapping Analysis of Mutant and Transductant Strains. Southernanalysis of HindIII digests of chromosomal DNA of various S43 strainsusing a Tn-specific probe (³²P-panel, 48 hr autoradiogram) reveals thatS43Tn7 (T) contains two Tn insertions (each Tn yields two bands due toan internal HindIII site). Transduction segregates the two Tns andproduces nonmucoid (N,N′) or mucoid (M,M′) colonies (two independentclones of each are shown). Wild-type S43 (W) DNA does not hybridize withthe probe. All the wild-type HindIII fragments detected with ethidiumbromide (EB panel) migrate as 10 kb (S; X HindIII standards in kb).Therefore, the chimeric Tn-tagged fragments (marked with arrows) werepurified and sequenced directly. An oligonucleotide probe specific forthe HA biosynthetic locus was derived from the fragment marked with thestar.

FIG. 6: Schematic Map of the HA Biosynthesis Locus and Various PlasmidConstructs. A restriction map of the complementing region of S43 DNA,containing two substantial ORFs, is shown. The hasA and hasB genes aretranslated in the same orientation but in different reading frames. Inthis schematic, the HasA open reading frame begins with the standard ATGcodon. Sites for EcORI (E), HindIII (H), ClaI (C), BglII (B), PstI (P),and EcoRV (R) are marked. The Tn insertion site was about 4 kb to theright of the E site on the wild-type map but the intervening chromosomalDNA was deleted in the 543Tn7 mutant (DeAngelis, et al., 1993). Thevarious pPD41 deletion constructs are depicted (black lines) below themap. The cross-hatched areas represent flanking sequences on either sideof the two open reading frames.

FIG. 7: Nucleotide and Deduced Protein Sequence of the HA Synthase gene,hasA. The DNA sequence surrounding the HA synthase ORF was determined onboth strands with Sequenase. The standard deduced start codon for aprotein (ATG) is indicated as the first amino acid in this figure. Thisputative start codon (ATG) is marked as position +1. Alternate startcodons (Gren, 1984) indicated in bold-face (GTG at −72 or TTG at −27 and−15) are present in-frame upstream from this ATG. The additional aminoacids comprising HasA, if alternative start codons are used, are shownin lower case. Hydrophobic stretches predicted to be membrane-associatedare underlined and Cys residues are shown stippled. The beginning ofHasB (Dougherty and van de Rijn, 1993) is also depicted at the lowerright. The sequence is in the GenBank database under Accession No.L20853.

FIG. 8: E. coli Minicell Analysis of pPD41 Deletion Constructs.Minicells from XI448 containing various plasmids were ³⁵S-labeled andthe proteins were separated on a 12.5% SDSPAGE gel. This autoradiogram(10 hr exposure) shows that when hasA or hasE genes are disrupted, thepredicted proteins (HasA, filled arrow at 42 kDa; HasB, open arrow at 45kDa) are likewise affected. The truncated versions of HasA (filledcircle) or HasB (open circle) are smaller as expected. Lanes: A, pATl9vector, B, pPD41L 7; C pPD41 5; D, pPD PstI; E, pPD EcoRV. Standards(BioRad, low MW) are marked in kDa.

FIG. 9: Buoyant density separation of acapsular and encapsulatedstreptococci. The procedure described herein in Example III was used inthis example to distinguish encapsulated, mucoid S43 cells andacapsular, nonmucoid NZI3I cells except that, for purposes ofillustration, the 65% Percoll layer was underlaid with a 100% Percollpad. Note that cells with a capsule (C) are at the top interface whilethe acapsular or enzyme-treated cells (A) appear at the lower interface(these cells would be in a pellet if not for the 100% Percoll pad)Hyaluronidase (HAase) treatment of the culture of the mucoid strainremoves the capsule and increases the density so that the majority ofthe cells appear in the pellet.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be exemplified herein in terms of preferredembodiments for the isolation and use of DNA segments comprisingsequences encoding the HA synthase gene from Streptococcal sources.However, it will be appreciated by those of skill in the art that inlight of the present disclosure the invention is also applicable to theisolation and use of the HA synthase enzyme from virtually any source,such as Streptococcus pyogenas, S. equisimilis, other group A or group Cstreptococcal strains or eukaryotic sources such as dermal or synovialfibroblasts, chondrocytes, trabecular-meshwork cells or rooster combmesodermal cells which contain HA synthase encoding DNA that is activelytranscribed (and is a suitable source of mRMA for the preparation ofcDNA libraries)

The preferred application of the present invention to the isolation anduse of streptococcal HA synthase DNA involves generally the steps of(Sambrook, et al., 1989) isolation of streptococcal genomic DNA;preparation of a genomic DNA library, such as in a bacteriophage lambda;screening the library with oligonucleotides from the derived sequence;isolating clones and subclones of phage with the oligonucleotide;excising the resident plasmid from within the phage genome or ligating apurified DNA into a selected site in a cloning vector; (TrieuCuot, etal., 1991) transfection of host Streptococcus or E. coli cells with therecombined vector; and selection of colonies expressing HA synthase orHA itself through the application of specially designed screeningprotocols. Following identification of a clone which contains the HAsynthase gene, one may desire to reengineer the HA synthase gene into apreferred host/vector/promoter system for enhanced production of HA.

A. Cloning of Hyaluronate Synthase Gene

To clone the HA synthase gene, hasA, the present inventors usedtransposon insertion mutagenesis to identify the locus that isresponsible for capsular formation. Genomic DNA from a transposon-taggedmutant of the Streptococcus pyogenes strain S43 was isolated frombacteria following hyaluronidase treatment, chloroform/isoamylextraction and ethanol precipitation. It is believed to be important toprovide a DNA fragment encoding a full length or essentially full lengthenzyme because the initial screening protocol requires expression of afunctional enzyme shown to synthesize HA.

The identification and verification of the HA synthase gene wasaccomplished by analyzing transposon-directed mutants and cells thathave been transformed with the HA synthase gene of the presentinvention. The assay is based on the formation of a polysaccharidecapsule in acapsular strains of Streptococci and heterologous bacteria.Initially TN916 insertional mutants were screened for an acapsular,non-mucoid phenotype. The ends of the transposon were then used toobtain sequence in the vicinity of the interrupted gene to direct thecloning of the wild-type hasA gene.

A contiguous three kilobase pair region of DNA (FIG. 1) was isolatedfrom Group A Streptococcus pyogenes [GAS] that can direct hyaluronicacid [HA] capsule biosynthesis in acapsular mutants as well asheterologous bacteria (FIG. 2). The DNA was identified by transposon 916insertional mutagenesis and subcloned into a plasmid shuttle vector.Mutant acapsular GAS or Entarococcus faecalis containing this plasmid,but not vector alone, displayed a mucoid phenotype on agar plates,possessed a capsule as seen by light microscopy, and produced HA inquantities comparable to wild-type GAS. The polysaccharide was shown tobe authentic HA based on its recognition by a specific HA-bindingproteoglycan and its degradation by Straptomyces hyaluronate lyase.Escherichia coli with the complementing plasmid also produced HA, but atonly 10% of the level made by the above cells. E. coli minicell analysisshowed that two proteins, 42 and 45 kDa, are expressed by the functionalDNA insert. Deletion analysis of the insert in the minicells revealedthat the 42 kDa protein is essential for HA production.

One may also desire to characterize the streptococcal or other HAsynthases in terms of their kinetics and physical and chemicalproperties. The parameters, Km and Vmax are determined from a doublereciprocal plot of the velocity of the reaction versus substrateconcentration (Lineweaver-Burke plot) Properties which may be ofinterest may include the enzyme's thermostability, optimum pH foractivity, effects of various ions, and effects of various inhibitors.Isoelectric focusing will be used to determine the isoelectric point ofthe synthase. Understanding these factors would provide basicinformation that may further allow one the ability to better determinewhat alterations in their primary sequence can provide additionaladvantages.

By appropriate modification of the DNA segment comprising the gene forHA synthase (e.g., deletion of membrane spanning domains of theprotein), the enzyme can be converted to a form that may be secreted bythe transfected bacterial host. This enzyme in soluble form, if stillactive in the ability to synthesize HA, would provide substantialimprovement in the ease of purification of this modified enzyme and inits potential utility in an enzyme reactor system for the in vitroproduction of HA.

B. Typical Genetic Engineering Methods Which May be Employed

If cells without formidable cell membrane barriers are used as hostcells, transfection is carried out by the calcium phosphateprecipitation method, well known to those of skill in the art (Sambrook,et al., 1989). However, other methods may also be used for introducingDNA into cells such as by nuclear injection, electroporation, protoplastfusion or by the Biolistic(tm) Bioparticle delivery System recentlydeveloped by DuPont (1989). The advantage of using this system is a hightransformation efficiency. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, the preferred method oftransfection is calcium treatment using calcium chloride (Sambrook, etal., 1989) or electroporation.

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and relegated in the formdesired to construct the plasmids required. Cleavage is performed bytreating with restriction enzyme (or enzymes) in suitable buffer. Ingeneral, about 1 μg plasmid or DNA fragments are used with about 1 unitof enzyme in about 20 μl of buffer solution. (Appropriate buffers andsubstrate amounts for particular restriction enzymes are specified bythe manufacturer.) Incubation times of about 1 hour at 37 degree(s) areworkable.

After incubations, protein is removed by extraction with phenol andchloroform, and the nucleic acid is recovered from the aqueous fractionby precipitation with ethanol. If blunt ends are required, thepreparation is treated for 15 minutes at 15 degree(s) C. with 10 unitsof Polymerase I (Klenow) phenol-chloroform extracted, and ethanolprecipitated. For ligation approximately equimolar amounts of thedesired components, suitably end tailored to provide correct matchingare treated with about 10 units T4 DNA ligase per 0.5 μg DNA. Whencleaved vectors are used as components, it may be useful to preventrelegation of the cleaved vector by pretreatment with bacterial alkalinephosphatase.

For analysis to confirm functional sequences in plasmids constructed,the ligation mixtures are used to transform E. coli K5strain Bi8337-41(Gupta, et al., 1982), and successful transformants selected byerythromycin resistance where appropriate. Plasmids from the library oftransformants are then screened for bacterial colonies that exhibit HAproduction. These colonies are picked, amplified and the plasmidspurified and analyzed by restriction mapping. The plasmids showingindications of a functional HA synthase gene are then furthercharacterized by sequence analysis by the method of Sanger (Sanger, etal., 1977), Messing (Messing, et al., 1981), or by the method of Maxam(Maxam, et al., 1980).

C. Host Cell Cultures and Vectors

In general, prokaryotes are preferred for the initial cloning of DNAsequences and construction of the vectors useful in the invention. It isanticipated that the best host cells may be Gram-positive cells,particularly those derived from the group A and group C Streptococcalstrains. Bacteria with a single membrane, but a thick cell wall such asStaphylococci and Streptococci are Gram-positive. Gram-negative bacteriasuch as E. coli contain two discrete membranes rather than onesurrounding the cell. Gram-negative organisms tend to have thinner cellwalls. The single membrane of the Gram-positive organisms is analogoustothe inner plasma membrane of Gramnegative bacteria. The preferred hostcells are Streptococcus strains that are mutated to become hyaluronidasenegative or otherwise inhibited (EP144019, EP266578, EP244757)Streptococcus strains that have been particularly useful as suitablehosts include S. pyoganas S43, S. equisimilis and S. zooepidamicus.

Although E. coli is Gram-negative it is, nonetheless, a useful host cellin many situations, as shown in Examples I and IV. E. coli SURE™ cellswere chosen as the initial recipient strain for transformation andcloning of the HA synthase gene because this strain has proven to bevery useful in recombinant DNA studies. It is a widely used host and isspecifically engineered for recombinant DNA work. E. coli X1448 waschosen for verification of HasA protein expression because of itsutility as a minicell expression system. Other E. coli strains may alsobe useful for expression of the shuttle vectors pATl9 and pSA3containing the HA synthase gene. For example, E. coli K12 strain 294(ATCC No. 31446) may be useful. Other strains which may be used includeE. coli B, and E. coli KS. These examples are, of course, intended to beillustrative rather than limiting.

Prokaryotes may also be used for expression. For the expression of HAsynthase in a form most likely to accommodate high molecular weight HAsynthesis, one may desire to employ Streptococcus species such as S.equisimilis, S. pyogenas or S. zooepidemicus. The aforementionedstrains, as well as E. coli W3110 (F-, lambda-prototrophic, ATCC Mo.273325), bacilli such as Bacillus subtilis, or other enterobacteriaceaesuch as Salmonella typhimurium or Serratia marcescens, and variousPsaudomonas species may also be used, as described in Example V.

In general, plasmid vectors containing origins of replication andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries an origin of replication, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species. pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. A pBR plasmid or a pUC plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

Those promoters most commonly used in recombinant DNA constructioninclude the lacZ promoter, tac promoter, the T7 bacteriophage promoter,B-lactamase (penicillinase) and tryptophan (trp) promoter system(Ausbel, et al., 1987). While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enabling askilled worker to ligate them functionally with plasmid vectors (Ausbel,et al., 1987). Also for use with the present invention one may utilizeintegration vectors.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomycas carevisiaa, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomycas, the plasmid YRp7, for example, is commonly used (Ausbel,et al. 1987). This plasmid already contains the trpl gene which providesa selection marker for a mutant strain of yeast lacking the ability togrow without tryptophan, for example ATCC No. 44076 or PEP4-l (Jones,1977). The presence of the trpl lesion as a characteristic of the yeasthost cell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Suitablepromoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes are also ligated into the expression vector3′ of the sequence desired to be expressed to provide polyadenylation ofthe RNA and termination. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions are thepromoter region for alcohol dehydrogenase 2, cytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Any plasmidvector containing a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (Tissue Culture, 1973) hasbecome a routine procedure in recent years (Sambrook, et al., 1989).Examples of such useful host cell lines are VERO and HeLa cells, Chinesehamster ovary (CHO) cell lines, and WI38, BHK, COS, and MDCK cell lines.

Other particularly useful host cell lines may be derived from dermal orsynovial fibroblasts, mesodermal cells of rooster comb or thetrabecular-meshwork cells of the eye. Expression vectors for such cellsordinarily include (if necessary) an origin of replication, a promoterlocated at the 5′ end of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsite, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, bovine papilloma virusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication.

Further, it is also possible, and often desirable, to utilize promoteror control sequences normally associated with the desired gene sequence,provided such control sequences are compatible with the host cellsystems. An origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter mechanismis often sufficient.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of theforegoing disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations which fall within the spiritand the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example I Isolation of the HA Synthase Locus

Tn insertional mutagenesis was used to tag and to mutate capsulebiosynthesis genes of GAS. The bacteriophage A25 transducing lysate fromone acapsular mutant, designated S43Tn7 and containing two Tn916elements, was found to transmit the nonmucoid phenotype to 3 out of Stransductants. The two Tn elements were segregated by transduction; oneTn insertion characterized by higher MW HindIII fragments was found onlyin the nonmucoid transductants (S43Td7N), while the other Tn insertionevent producing lower MW fragments was found only in the mucoid cells(S43Td7M). Nonmucoid transductants did not possess HA synthase activityor a capsule as determined by enzyme assays of membranes or by lightmicroscopy, respectively.

Materials and Methods

Materials: Media reagents were from Difco. Restriction and DNA modifyingenzymes were from Promega unless otherwise noted. Syntheticoligonucleotides were made at the UTMB Synthesis Facility or by KeystoneLaboratories. All other reagents were of the highest grade available andfrom Sigma except where stated otherwise.

Strains and Vectors: Escherichia coli was maintained on LB and grown inSuperbroth with antibiotics for plasmid production. Other bacteria weregrown as standing cultures on Todd-Hewitt broth supplemented with 1%yeast [THY] and horse serum (5-10%, Gibco). Cultures to be assayed forHA were grown using the dialyzate from dialyzed THY broth, (i.e.,nutrients <10-14 kDa). The mucoid GAS strain, S43/192/4, was obtainedfrom the Rockefeller Collection (Dochez, et al., 1919). Spontaneousstrep^(r) strains used as Tn acceptors were selected by plating ˜10⁹cells on THY plates containing 1 mg/ml streptomycin and 3% defibrinatedsheep blood (Colorado Serum). Entarococcus faecalis CG110, a Tn9?6 donor(tetracycline resistant, 5 p/ml), and PAM118, a plasmid with a Tn916insert, were generously supplied by D. Clewell (Gawron-Burke andClewell, 1984). The E. coli/Gram-positive shuttle vector pATl9(erythromycin resistant, 8 μg/ml for Gram-positive or 150-200 μg/ml forE. coli) was provided by P. Courvalin (Trieu-Cuot, et al., 1991). The E.faecalis host strain OG1RF was obtained from G. Dunny (Dunny, et al.,1991). E. coli minicell strain X1448 (Meagher, et al., 1977) wassupplied by R. M. Macnab. The E. coli hosts used were SURE, XL1-Blue(Stratagene), LE392, and KW251 (Promega).

DNA Purification and Sequencing: Streptococcal chromosomal DNA wasobtained by the method of Caparon and Scott (Caparon and Scott, 1991).E. coli plasmid DNA was purified by the Instaprep method (5 Prime→3Prime, Inc.) for screening or by the SDS/alkali method for cloning andblotting procedures (Sambrook, et al., 1989). Agarose (BioRad)gel-isolated DNA <7 kb was purified by GeneClean (Bio 101), while longerfragments were isolated using GlassMax cartridges (Gibco) to minimizeshearing. λ DNA was prepared from phage purified on glycerol gradients(Sambrook, et al., 1989). Sequencing of double stranded plasmids wasperformed with Sequenase 2.0 (US Biochem) and α[³⁵S]thiodATP (Amersham)

Lambda Library Production: The λXZAPII library (Stratagene, 1-10 kbcapacity) contained S43 wild-type DNA digested extensively with EcoRI.The λGEM system inserts (Promega, 9-23 kb capacity) consisted of S43 DNApartially digested with Sau3A. The λZAP system inserts from selectedphage were excised and converted to plasmid form by coinfection with M13helper phage (R408 or Exassist) according to Stratagene protocols.

Transposon Mutagenesis and Mutant Selection: The detailed methods for Tnmutagenesis, mutant selection, and the isolation and characterization ofthe Tn-tagged DNA are described in Example 3. Briefly, Tn insertionalmutagenesis was done on a mucoid strep^(r) S43 strain using the methodof O'Connor and Cleary (O'Connor and Cleary, 1987). The nonmucoid mutantcells were enriched by Percoll step gradients in analogy to work donewith Group B Streptococci (H{dot over (a)}kansson and Holm, 1986) afterovernight outgrowth in double selective media as in Example III and FIG.9. Candidate mutants of capsule biosynthesis were picked by visuallyscreening for dry, discrete colonies versus wild-type wet, spreadingcolonies. The nonmucoid strains chosen for study did not a) float in 50%Percoll, b) produce a capsule visualizable by India ink exclusion bylight microscopy (Collins and Lyne, 1976), c) possess detectable HAsynthase activity in membrane preparations, or d) synthesizeextracellular HA as determined by a sensitive HA assay (see HAPolysaccharide Analysis). Transduction with the streptococcal phage A25(kindly supplied by M. Caparon) was used to determine the relevance ofthe various Tn916 insertions in the nonmucoid strains (Caparon andScott, 1991).

Transposon Mapping and Isolation of the HA Synthesis Locus: An overviewof the isolation procedures, as described herein below in Example 3, andas follows. After electrophoresis of chromosomal HindIII digests, theagarose gels (0.5-0.6%) were dried down directly (Ehtesham and Hasnain,1991) and probed with the Tn916-containing EcORI fragment of pAMll8labeled by the random primer method (Sambrook, et al., 1989). It wasnoted that one of the Tn916/S43 chimeric DNA fragments consistentlymigrated slower than the other fragments in HindIII digests of wild-typeDNA. This chimeric fragment from preparative digests (5-15 μg) wasisolated from an agarose gel with GlassMax and used as a DNA sequencingtemplate to determine the sequence of the junction at the site ofinsertion; the DNA was not cloned first. A synthetic oligonucleotidederived from the termini of the right HindIII fragment of Tn916(Clewell, et al., 1988) that reads outward from the Tn(AAAGTGTGATAAGTCC) (SEQ ID NO:4) was employed as a primer in amodification of the Sequenase method (US Biochem) for plasmids. Theintact wild-type DNA was then obtained by screening the lambda librariesin the typical fashion (Sambrook, et al., 1989) with end-labeledoligonucleotide (TGGCACAATATGTCAGCCC) (SEQ ID NO:5) derived from thechromosomal sequence determined as above.

The HA biosynthesis locus is very unstable with respect to DNA deletion.The present inventors found that this characteristic made the subcloningdifficult. The inventors found that three factors were essential inorder to obtain stable clones of the hasA locus in its entirety: (1) useof a recombination deficient host (e.g., E. coli SURE™ cells), (2) useof the electroporation method of transformation and, (3) performing therecovery and all further growth of the recombinant cells at 30°-32° C.If these conditions were not followed for the subcloning of the hasADNA, the vast majority of the target insert DNA was lost. Additionally,during the routine subculture of pPD41A5 at 37° C., the inventors havenoted that deleted plasmids arose at a high rate. Therefore, atemperature of about 30° C. was used for any applications in which hasAand plasmid integrity was concerned.

To create a deletion set, the pPD41 plasmid linearized with XbaI andSphI was truncated by limited Exonuclease III digestion and Mung Beannuclease treatment according to the Stratagene kit. The ligationmixtures were transformed into Epicurean SURE cells (Stratagene) andscreened for insert size. E. coli was also transformed by the Ca²⁺method (Sambrook, et al., 1989).

Results

The gel-purified, Tn-tagged chromosomal DNA from S43Tn7 was useddirectly as a template in sequencing reactions with a Tn-specific primerthat reads outward from the Tn terminus and into the insertion site. Anoligonucleotide probe corresponding to a portion of the sequence of theinterrupted DNA associated with the nonmucoid phenotype was then used asa hybridization probe for screening wild-type S43 genomic DNA librariesin phage. An excised ZAP clone, designated pB3, containing a 5.5 kbEcORI fragment was obtained (FIG. 1). However, differences between thewild-type and Tn-mutant genomes were noted by Southern analysis. Sinceprevious studies showed that Tn916 can cause deletions of chromosomalDNA (Dougherty and van de Rijn, 1992), the inventors then determinedthat at least 4 kb of DNA flanking the small arm (5 kb HindIII portionof Tn) was missing in S43Tn7 and S43Td7N (FIG. 1). Therefore, a largerwild-type genomic fragment spanning the deletion was obtained from theGEM library. A 6.6 kb portion of DNA adjacent to pB3 was subcloned intopATl9 and designated pPD41 (FIG. 1).

The various plasmids were introduced into acapsular Streptococcusmutants as well as into heterologous bacteria. Table II shows HAproduced by cultures (pooled spent media plus SDS cell extract) grown indTHY. HA was determined by the HA TEST assay kit (Pharmacia, Uppsala,Sweden). The HA concentration was normalized per A600 unit of cells. ThepPD41 plasmid confers the ability to synthesize HA in the three speciestested (DeAngelis, et al., 1993a). The truncated version, pPD41A5, wasthe minimal size functional plasmid obtained.

TABLE II HA production by various constructs. BACTERIAL HA STRAINPLASMID ng/μl dTHY media alone —    1¹ S. pyogenas S43 — 1120 S43Tn7pAT19   8 S43Tn7 pPD41  640 S43Trill pAT19   3 S43Tn11 pPD4I  860S43Tn11 pB3   6 E. faecalis OG1RF pAT19   2 pPD41  690 E. coli SUREpPD4I  60 pPD4IΔ4  80 pPD41ΔS  80 pPD41Δ6    2² ¹The media alonecontains about 1 ng/μl HA after dialysis. The apparent “background” isreported for each host. ²S43Tn11 is an acapsular, HA synthase negative,Tn-containing strain that was shown by transduction analysis to be aspontaneous mutant.

Example II Characterization of the HA Synthase Locus

When pPD41 was electroporated into the original acapsular Tn mutant,S43Tn7, or a spontaneously arising nonmucoid strain, S43Tnll,transformant colonies displayed the mucoid phenotype on agar plates,while cells with pB3 or pAT19 were nonmucoid. The capsules of thecomplemented cells were easily visualized by microscopy with India inkand were indistinguishable from the wild-type (FIG. 2). Ovine testicularhyaluronidase treatment of the cultures completely destroyed thesecapsules.

Using a sensitive radiometric assay, HA was detected in the cultures ofthe Tn-mutants containing pPD41 in amounts comparable to the wild-typeparent (as shown in Table II). Transformants with p33 or pAT19, as wellas the original mutant without plasmid, did not produce HA (Table II).The HA was detected by proteoglycan binding; this high affinityinteraction is very specific and is widely accepted as evidence for thepresence of HA (Tengblad, 1980). As in the case of the GAS mutants, E.feacalis or E. coli containing pPD41 produced HA (Table 1). Bymicroscopy with India ink, E. faecalis, but not E. coli, containingpPD41 possessed a substantial capsule.

Materials and Methods

HA Synthase Preparation and Assay: HA synthase was obtained frommembranes of late log phase cells disrupted by sonication in PBS (20%cell suspension, dry ice/50% methanol bath, 4×2 min, Heat Systems W-380with microprobe). Debris was removed by centrifugation at 12,000 g×10min at 4° C., and then the membrane fraction was harvested from thesupernatant by ultracentrifugation (100,000 g×60 min. The membranes(5-300 μg protein) were incubated with UDP-[¹⁴C]G1cUA (250 mCi/mMole,1CN) in the assay as described by Triscott and van de Rijn (Triscott andvan de Rijn, 1986). Specificity of polymerization was tested by omittingUDP-GlcNAc. Incorporation of the radiolabel into high MW polymers wasmeasured by paper chromatography (Prehm, 1983).

HA Polysaccharide Analysis: The presence of HA in bacterial cultures(early log for S43 derivatives, late log for all others) was determinedusing the HA TEST radiometric assay (range 5-500 ng Pharmacia). Thedetection is based on inhibition of ¹²⁵I proteoglycan binding to HAimmobilized on beads by soluble HA in the sample. Secreted or releasedHA in cultures grown on dTHY was measured by assay of the supernatantfraction after centrifugation (1,000 g×5 min). Cell-associated HA wasdetermined by extracting the cell pellet in 1/10 vol of PBS containing0.01% SDS for 40 min at 37° C. The cells were then removed bycentrifugation as above. The final SDS concentration in the HA assaynever exceeded 0.001%.

HA was also purified by cetyltrimethylammonium bromide (CTAB)precipitation. The pooled media supernatant and cell extracts (treatedwith 0.1 mg/ml trypsin for 40 min at 37° C. followed by SDS extractionas above) from 25 ml cultures were adjusted to 0.3% CTAB and allowed tostand at 37° C. for 15 min. The precipitate was collected bycentrifugation (3,000 g×20 min at room temperature) and resuspended in0.7 ml of 2 M NaCl with gentle mixing for 40 min. The solubilizedfraction (clarified by high speed centrifugation, 1,000 g×5 min) wasthen precipitated by addition of 2 volumes of ethanol. After 5 min, thesolids were collected by high speed centrifugation. The pellet waswashed with 70% ethanol/30% 2 M NaCl and then 70% ethanol. After briefdrying, the pellet was resuspended in 2 M NaCl and ethanol precipitationwas repeated as above. The final pellet was dissolved in 0.2 ml waterovernight with gentle mixing at 4° C.

The uronic acid content of the purified material was measured by thecarbazole assay with glucuronic acid as standard (Bitter and Muir,1962). The MW of the polymers and authentic rooster comb HA standards(Lifecore) were compared by polyacrylamide gel electrophoresis (PAGE)(Min and Cowman, 1986) and size exclusion chromatography. To verify thenature of the polysaccharide, samples were digested with hyaluronatelyase from Streptomycas hyaluroniticus in 50 mM sodium acetate, pH 5.3,at 42° C. before analysis. Sepharose 4B (Pharmacia, 1×25 cm column, 20ml bed volume) eluted with PBS was used to fractionate the variouspolymers. The column fractions were assayed by the carbazole method andthe HA TEST kit was used to confirm the major peak identity. The columnwas calibrated with dextrans (2×10⁶, 5×10⁵, 4×10⁴ Da; Pharmacia) andlactose as well as the HA standards.

Minicell Analysis: The identity of plasmid-encoded polypeptides wasdetermined by radiolabeling proteins produced in minicells (Matsumura,et al., 1977). Minicells from E. coli χ1448 containing pAT19, pPD41ΔS,or pPD41Δ7 were harvested from sucrose gradients and washed with PBScontaining 0.01% gelatin. The minicells were incubated at 37° C. for 1hr in minimal salts with glycerol and all amino acids except Met andCys. The minicells were then labeled with ³⁵S-Translabel (1CN) for 30min at 37° C. followed by a 5 min Met/Cys chase. The minicells were thenwashed with PBS and analyzed by SDS-PAGE after boiling (3 min) inLaemmli sample buffer (Laemmli, 1970). The gels were stained withCoomassie Blue, dried, and exposed to XAR-S film (Kodak).

Miscellaneous: Of several published electroporation methods for use withGram-positive bacteria, the present inventors found that only thetechnique of Caparon and Scott (Caparon and Scott, 1991) was successfulin transforming S43 derivatives with plasmids (0.5 to 20transformants/μg DNA).

Results

To determine the minimum size of the locus directing HA biosynthesis,the complementing DNA insert of pPD41 was reduced by limited exonucleasedigestion of the plasmid from the SacI end of the pAT19 multiple cloningsite. E. coli transformed with plasmids containing an insert of ˜3 kb(e.g., 5 min deletion, pPD41Δ5; see FIG. 1) still produced HA, whilecells with smaller inserts (e.g., 6 min deletion, pPD41Δ6, ˜2.3 kb or 7min deletion, pPD41Δ7, ˜1.7 kb) did not make HA (Table II). The E.faecalis cells transformed with pPD41A5 produced hyaluronidase-sensitivecapsules as assessed by microscopy (FIGS. 2D & 2F), and formed mucoidcolonies on agar plates, whereas the cells containing pPD41Δ7 wereequivalent to untransformed E. faecalis (FIG. 2E). E. faecais has notbeen reported to produce a capsule or any exopolysaccharides. Therefore,the pPD41Δ5 insert is responsible for HA capsule biosynthesis.

The extracellular polysaccharides produced by the various bacteriacontaining the pPD41 family of plasmids were further characterized bygel filtration chromatography and PAGE. All polysaccharides possessedM_(r)s on the order of 10⁶, since they eluted in the void volume and thefirst included fractions on the Sepharose 4B column, well before a 500kDa dextran standard (not shown). By electrophoretic analysis, wild-typeS43 HA and polysaccharide from mutant GAS strains with pPD41 or E.faecais with pPD41Δ5 migrated similarly compared to authentic high MW HAstandards (FIG. 3). The specific Straptomyces hyaluronate lyase degradedboth authentic HA and the polysaccharides produced by S43Tn7 or E.faecalis containing the complementing plasmids (FIG. 3)

The E. coli minicell system provides a convenient way to determine thenumber and size of proteins encoded by genes on episomal plasmids(Meagher, et al., 1977; Matsumura, et al., 1977). Minicell analysisrevealed that at least two proteins were encoded on the complementingDNA that directed HA capsule biosynthesis (FIG. 4). In addition tovector-derived proteins, the nonfunctional pPD41A7 encoded a prominent45-kDa protein. Minicells with the HA-producing pPD41Δ5 plasmid produceda 42-kDa protein as well as the 45-kDa species, indicating that theformer protein is essential for HA synthesis. The present inventorscalculate that about 80% of the coding capacity of the ˜3 kb insert inpPD41Δ5 is utilized for these two proteins.

Neither the purification nor the cloning of the HA synthase has beensuccessfully demonstrated in either bacteria or eukaryotes. Prehm andMausolf (Prehm and Mausolf, 1986) implicated a 52-kDa protein from GCSas the HA synthase by affinity labeling with periodate-oxidized sugarnucleotides. A polyclonal antibody to this protein inhibited HA synthaseactivity of membranes (Prehm and Mausolf, 1986). However, the active HAsynthase was not purified. The gene corresponding to the 52 kDa proteinwas then cloned using the antibody and, although the assertion ofcloning the HA synthase gene was made, the present inventors have foundthis conclusion to be invalid; the deduced sequence had similarities toan oligopeptide transport protein of Bacillus subtilis (Lansing, et al.,1993). van de Rijn and Drake (van de Rijn and Drake, 1992) found threepolypeptides (42, 33, and 27 kDa) from GAS and GCS membranes that werephotoaffinity labeled by a substrate analogue, azido UDPglucuronic acid.Excess UDP-G1cA inhibited incorporation of the analogue but labeling ofall three polypeptides was stimulated upon addition of the otherprecursor of HA, UDP-GlcNAc (van de Rijn and Drake, 1992). The 42 kDaprotein labeled in the pPD41A5-containing minicells is the same size asone of the proteins photoaffinity labeled with a substrate analog (vande Rijn and Drake, 1992). However, the proteins of 32 and 27 kDa thatwere also labeled were not observed in the inventors studies.

Dougherty and van de Rijn (Dougherty and van de Rijn, 1992) used Tninsertional mutagenesis to identify a GAS genetic locus associated withHA synthase activity. Two open reading frames were describedschematically but no sequence information was reported and no in vivo orin vitro HA synthase activity was reported.

None of the above studies functionally reconstituted HA synthesis in anacapsular mutant or in heterologous bacteria with cloned streptococcalDNA. The results of the inventors, however, show that a contiguous 3 kbregion of the GAS chromosome, encoding proteins of 42 and 45 kDa, candirect HA biosynthesis in GAS mutants as well as in E. faecalis andGram-negative E. coli.

Example III Cloning of the HA Synthase Gene

The HA synthase gene of GAS was initially identified by Tn insertionalmutagenesis as described in Example I. The bacteriophage A25 transducinglysate (Caparon and Scott, 1991) from one acapsular mutant (designatedS43Tn7), which contained two Tn elements, transmitted the nonmucoidphenotype to 3 out of S transductants (FIG. 5). The nonmucoidtransductants did not possess HA synthase activity or a capsule by lightmicroscopy, but the mucoid transductants were equivalent to wild-typeS43. HindIII digests of mutant S43Tn7 chromosomal DNA showed two bandsmigrating at 16 and 18 kb on agarose gels that corresponded to thehigher MW bands detected by a Tn-specific probe on Southern blots of allTN916 mutants (FIG. 5) These larger Species are the result of adding 10kb of Tn DNA to the S43 HindIII fragment at the insertion site.

Since the Tn-tagged DNA from S43Tn7 was well resolved from the otherHindIII fragments, it could be gel-purified. The 18 kb chimeric fragmentassociated with the HA biosynthesis defect was therefore used directlyas a template for sequencing reactions with a Tn-specific primer thatreads outward from the Tn terminus and into the interrupted gene. Anoligonucleotide (SEQ ID NO:5), corresponding to a portion of thesequence of the interrupted gene from the 18 kb chimeric fragment, wasused as a hybridization probe for screening wild-type S43 genomic DNAlibraries in λphage.

An excised λZAP clone, pB3, containing a S.S kb EcoRI fragment wasselected and studied further. However, Southern analysis utilizingvarious oligonucleotide probes to the sequence of pB3 revealed somediscrepancies between the wild-type and Tn-mutant genomes (e.g., the SEQID NO:6 oligo hybridized to S43 but not S43Tn7, while the SEQ ID NO: 5oligo hybridized to both). Therefore, a larger genomic fragment spanningthe Tn-induced deletion (DeAngelis, et al., 1993a,b) was obtained fromthe λGEM library. After an extensive subcloning effort and subsequentexonuclease III deletion, a 3.2 kb fragment of S43 DNA was identified asa locus that could direct HA biosynthesis (DeAngelis, et al., 1993a)

Materials and Methods

Materials and Strains: Restriction and DNA modifying enzymes were fromPromega unless otherwise noted. All other reagents were of the highestgrade available from Sigma unless stated otherwise. Media reagents werefrom Difcco Cultures to be assayed for HA were grown using the dialysatefrom dialyzed THY broth (i.e., nutrients <10-14 kDa). The mucoid GASstrain, S43/192/4, was obtained from the Rockefeller Collection (Dochez,et al., 1919). E. coli K5 (Bi8337-41) was obtained from I. and F. Orskov(Copenhagen, Denmark; Gupta, et al., 1982). All other strains andplasmids used were described by DeAngelis, et al., (1993a).

Tn Mutagenesis and Mutant Selection: Tn insertional mutagenesis wasconducted by the method of O'Connor and Cleary (O'Connor and Cleary,1987) except that ovine hyaluronidase (Type V) was added to the GASculture (0.2 mg/ml, 1 hr at 37° C.) after overnight growth and used at ahigher concentration (0.1 mg/ml) in the mating plate media. The Tn916donor, Enterococcus faecalis CG110 (Gawron-Burke and Clewell, 1984), wasmated on nitrocellulose filters (88 mm, 0.45 μm, Micron Separation,Inc.) with strep^(r) S43. The mating mixture was scraped off the filterswith 0.4 ml THY containing, 1 mg/ml streptomycin and S jig/mltetracycline.

The nonmucoid mutant cells were then enriched over Percoll (Pharmacia)step gradients (DeAngelis, et al., 1993b; H{dot over (a)}kansson andHolm, 1986) as illustrated in FIG. 9. This selection process allowedabout a thousand-fold more bacteria to be more readily examined than ifplating methods were used directly after the mating step. Acapsular (orhyaluronidase-treated) cells pellet through 50% Percoll, but mucoidcells float at the interface. After overnight outgrowth (50-70 μl matingmixture/5 ml double selective media with 5% serum in a 15 ml tube), thecultures were underlaid with 2 ml of 50% Percoll in water andcentrifuged (3,000 g×10 min). The media, the cells at the interface, andmost of the Percoll were removed by aspiration and the high density“cell pellet” fraction was then used to inoculate 5 ml of fresh doubleselective media. Two further rounds of outgrowth for 4-8 hrs(A₆₀₀=0.2-0.6) and gradient enrichment were performed. Portions of thefinal cell pellet were streaked on double selective plates containing 5%sheep blood and visually screened for candidate mutants of capsulebiosynthesis: those with dry, discrete colonies versus wild-type wet,spreading colonies.

The mutants were streak-purified and verified to be similar to wild-type543 with respect to vigor, B-hemolysis, DNase secretion (usingDNA/methyl green agar), and production of Group A carbohydrate(Ventrescreen, Hycor). Thirteen strains did not have HA synthaseactivity, produce capsules or contain HA (DeAngelis, et al., 1993a) butonly one strain, S43Tn7, transduced (Caparon and Scott, 1991) thenonmucoid phenotype.

Tn Mapping and Gene Isolation: Chromosomal DNA purified (Caparon andScott, 1991) from the mutants and transductants was cut with HindIII andanalyzed by Southern hybridization. After electrophoresis, the agarosegels were dried down directly (Ehtesham and Hasnain, 1991) and probedwith the Tn-containing EcORI fragment of pAMll8 (Gawron-Burke andClewell, 1984) labeled by random priming (New England Biolab kit). Thehybridization was conducted overnight at 65° C. in 1×HPB (0.5 M NaCl, 5mM EDTA, 0.1 N Na₂PO₄, pH 7.2) containing 1% sarcosyl, and the gel wasthen washed for 40 min with 20 mM Tris HCl, pH 8 at 22° C.

The 16 or 18 kb chimeric Tn-tagged fragments from preparative digests(5-15 μg) of S43Tn7 were isolated from gel slices using GlassMAX (LifeTechnologies) according to the manufacturer's instructions except thatthe DNA was eluted from the GlassMAX unit with 3 sequential additions ofwater at 65° C. The Sequenase method (USBiochem) for plasmids, withmodifications noted below, was employed to sequence the junction at thesite of Tn insertion directly from chromosomal DNA. A syntheticoligonucleotide (AAGTGTGATAAGTCC) (SEQ ID NO:4) based on the termini ofthe right arm of TN916 that reads outward into the interrupted gene wasused as the primer (Clewell, et al., 1988).

The purified DNA fragment (50-100 ng) was denatured with NaOH,neutralized with sodium acetate, and quickly ethanol precipitated in thepresence of 10 μg of phenol/CH₃C1 extracted glycogen. The primer SEQ IDNO:4 (0.22 μmol) was annealed to the template by slow cooling from 65°to 30° C. The labeling phase of the reaction was done with Mn² buffer,1:15 diluted labeling mix, and α[³⁵S]thiodATP (Amersham, 3,000 Ci/mMol)for 2 min at 20° C. The termination phase was done for 5 min at 37° C.with extension mix in the A and T reactions (0.6 μl) due to the A/Trichnature of streptococcal DNA. Gels were electrophoresed, processed(Sambrook, et al., 1989), and exposed to XAR-S film for 1-10 days atroom temperature. Typically, the sequence of the Tn terminus/junction(6-10 bp) and 20-40 bases of the adjacent tagged streptococcal DNA wereobtained.

The oligonucleotide derived from the chromosomal sequence determinedabove was used to screen two lambda libraries (DeAngelis, et al.,1993a,b) to obtain the intact wild-type DNA, in which the Tn insertionhad occurred in the mutant. The phage were adsorbed onto nitrocellulosefilters and processed in the typical fashion (Sambrook et al., 1989).The filters were hybridized with end-labeled oligonucleotide5′-TGGCACAATATGTCAGCCC-3′ (SEQ ID NO: 5), in 18×HPB (1 pmol probe/8 ml)with 1% sarcosyl, 0.5% nonfat milk at 42° C. for 3 hr. and washed with0.5×HPB at the same temperature for 1 hr. The plaques yielding thestrongest signal were replated and rescreened twice. Purified phage froma λZAP library were converted to plasmid form by coinfection of SURE orSOLR cells (Stratagene) with the Exassist helper phage (Stratagene). Oneclone, pB3, was analyzed by sequencing with Sequenase using the standardprotocols. The phage selected from the λGEM library using SEQ ID NO:5were screened with another oligonucleotide (5′-TATGGCTTAGTGCCATTCG-3′)(SEQ ID NO:6), corresponding to the sequence found near the end of thepB3 insert, in order to obtain DNA adjacent to pB3.

Two positively hybridizing λGEM isolates, which formed small plaques andgrew poorly in liquid lysates, were obtained. Large scale plate lysateswith top and bottom agarose were needed in order to prepare their DNA(Sambrook, et al., 1989). The two clones (λ1x and λ2Y with 20 and 12 kbinserts, respectively) contained the same region of DNA as determined bydirect sequencing of the X DNA insert using the Circumvent method (NewEngland Biolabs) and end-labeled oligonucleotide SEQ ID NO:6. Thesequence obtained beyond the EcoRI site of pB3 (left site; DeAngelis, etal., 1993a) was used to make another oligonucleotide(5′-CAATCATACCACCAACTGC-3′) (SEQ ID NO:7), for mapping analysis of the λclones treated with various restriction enzymes.

Southern blot analysis showed that a fragment of about 7 kb could beexcised from the smaller λ2Y clone using the EcoRI site in the S43 DNAand the SacI site of the λ vector. A portion of the digest was purifiedwith a Magic minicolumn (Promega) and the fragments were ligated topAT19 shuttle vector (Trieu-Cuot, et al., 1991) digested with EcoRI andSstI (Life Technologies). Attempts to subclone the streptococcalfragment in its entirety were thwarted by spontaneous deletions upontransformation into E. coli JM109. After using Epicurean competent SUREcells (Stratagene), using 32° C. for transformation recovery and allfurther growth, and restriction mapping ˜70 recombinant colonies, aclone containing a 6.6 kb insert, designated pPD41, was obtained thatcould complement the HA biosynthesis defect of mutant GAS (DeAngelis, etal., 1993a).

Results Isolation of Mutants

The inventors have used the difference in relative buoyant density toisolate acapsular mutants from a mating mixture of Streptococcuspyogenes strain S43 and an Enterococcus faecalis transposon TN916 donor(DeAngelis et al., 1993b). After centrifugation over a simple Percollstep gradient (50% pads), the cell pellet was harvested and used as aculture inoculum. Repeated cycles of growth and separation on Percollgradients were performed to enrich for acapsular mutants in order toavoid painstakingly screening hundreds of plates inoculated withunselected cultures. The initial cell “pellet” may not be visible, butif it is processed for 3 additional cycles (after aspiration of thesupernatant) even spontaneous acapsular mutants that occur at lowfrequency are obtained. Conversely, mucoid varieties can be enrichedfrom mixtures of both cell-types or spontaneous revertants of nonmucoidmutants can be recovered; in this case the interface is harvested with apipette and repeatedly processed as above. Isolation of either phenotypeis finally accomplished by streaking out on agar plates.

Quantitation of the capsular and acapsular phenotypes in a population ofbacteria may be obtained by measuring the ratio of relative A₆₀₀ ofresuspended cells harvested from both locations in the step gradient.This method is broadly applicable to other encapsulated microorganismsbesides Group A Streptococci (e.g., Group C; unpublished observation)but optimization of the Percoll concentration and capsule degradingreaction conditions may be necessary.

The utility and optimization of the buoyant density centrifugationtechnique was also studied with various encapsulated and acapsularstrains. The inventors have determined optimal concentrations of Percollfor separating encapsulated cells and acapsular cells by first usingdiscontinuous step gradients of Percoll (e.g., 50%, 63%, 75%, 87%).Wild-type S43 cells were found at the 50%/medium interface.Hyaluronidase-treated wild-type S43 cells collected at the 63%/75%Percoll interface near the green marker beads (DMB-7, 1.10 mg/ml). Thisdensity value was close to the measured value reported as the “typical”bacterial cell density (1.08 mg/ml) in a recent survey (Guerrero, etal., 1985). Light microscopy with India ink (Collins and Lyne, 1976) wasused to examine bacteria at both positions after centrifugation; all thecells at the medium/50% interface possessed capsules, while the cellswith the highest density at the 630%/750% interface had no detectablecapsule.

For bacteria with, smaller capsules than the highly mucoid S43 strain, asimple 65% Percoll pad could distinguish capsule phenotypes among anarray of Group A streptococci strains. Nonencapsulated cells were foundin the pellet, while encapsulated cells appeared at the interfacebetween the yellow media and clear Percoll (FIG. 9). The inventorsdetermined the presence or absence of HA on the various strains by usinga sensitive radiometric assay with a detection limit of 0.4 μg/ml ofculture media. If the bacteria made at least 7 μg of HA per ml of cellsper 1 A₆₀₀ unit, they were found at the interface of the Percoll layerand media. One strain, DW 1009, that produced 4 μg of HA per ml ofcells, however, appeared in the pellet and, therefore, appears to bebelow the limit of detection by our simple and rapid centrifugationmethod. This strain, however, showed nonmucoid colony morphology and noevidence of a capsule by light microscopy. Hyaluronidase-treatment ofthe cultures of mucoid strains before centrifugation caused the vastmajority of cells to pellet (FIG. 9), although some small clumps ofcells may remain at the medium/Percoll interface due to incompletedigestion of their capsule.

The density separation method is surprisingly sensitive to the bacterialHA level. Strain NSA156 produced 7 μg/ml HA and floated on 65% Percoll.This strain, which produces only about 0.5% to 4% of the HA made by mostencapsulated strains, does not appear mucoid on plates and its capsulewas not visible with the light microscope. A further asset of thepresent method is that very small amounts of cells are readily visible;cells at the 65% Percoll/media interface, when viewed at an angle, cloudthe junction's usual “mirror-like” appearance, while higher densitycells are concentrated by the conical bottom of the centrifuge tube.Another benefit of this method is that it circumvents the need for fresh(<2 month old) radiometric assay kits for detection of hyaluronic acidand, therefore, such kits do not need to be continuously available inthe laboratory. If further quantitation is needed on selected strains,these can also be stored and tested at a later date.

This density separation method is well suited for the sensitivedetermination of the presence of an HA capsule in clinical streptococciisolates; bacitracin-sensitive, B-hemolytic colonies from standard bloodplates can be picked and assayed. In light of the capsule's importanceas a virulence factor and the resurgence of streptococcal diseases inthe USA and Europe, monitoring HA capsule production may be useful fortracking virulent strains or epidemiological trends. The only equipmentneeded is a 37° C. incubator or waterbath and a low-speed clinicalcentrifuge. In less than one day after the initial streak isolation onan agar plate, and with only a few minutes of hands on labor, manyisolates can be screened for capsule production. Inclusion of thehyaluronidase-treated control tube may not be necessary during routinescreening, but rather can be used subsequently for verification of HAproduction.

hasA Cloning

The sequence of the complementing streptococcal DNA, the insert ofpPD41Δ5, was obtained using both the nested nuclease deletion set withthe M13 vector primers and the functional plasmid with customoligonucleotides. Two major ORFs were present (FIG. 6) in agreement withthe earlier minicell analysis (DeAngelis, et al., 1993a). The sequenceof the first ORF, hasA, reveals the primary structure of a previouslyundescribed protein (FIG. 7) (DeAngelis, et al. (1993b). The deducedpolypeptide contains 395 residues with a M^(r)=45,063 if the standardATG initiation codon is used (or up to 419 residues if the alternate GTGinitiation codon at position −72 is used). The 42-kDa protein observedby SDS-PAGE analysis of pPD41ΔS minicells is assigned to be NasA becausethe pPD41Δ7 plasmid, missing about half of the hasA gene (FIG. 6), doesnot produce the 42 kDa species (FIG. 8). HasA is predicted (Kyte andDoolittle, 1982) to be an integral membrane protein due to at least fourmembrane-associated regions (3 predicted transmembrane segments) and tohave a pI of 8.2.

Example IV Further Characterization of the hasA Gene

To identify the role of the two genes on the complementing streptococcalDNA, two constructs were made that substantially truncated either hasAor hasB (FIG. 6). One plasmid, pPDΔEcoRV, should produce the intact45-kDa protein, HasB. The other, pPDiΔPstI, should make the intact42-kDa protein, HasA. The pPDΔEcoRV construct, in which the truncatedHasA gene produced a new 27-kDa species (instead of the 42-kDa protein)as determined in minicells (FIG. 8), did not confer the ability toproduce HA in any host (Table III).

Materials and Methods

Polypeptides encoded by plasmid genes were identified by ³⁵S-labeling ofproteins produced in minicells from E. coli X1448 (Meagher, et al.,1977), containing pAT19 alone or various constructs containing S43 DNA,as described by DeAngelis (DeAngelis, et al., 1993a). DNA purificationand sequencing, lambda library production, nested deletion setconstruction, and HA synthase preparation and assay were performed asdescribed earlier (DeAngelis, et al., 1993a). Targeted internaldeletions were made by digesting pPD41Δ5 DNA with either EcORV or PstI,purifying the DNA with Magic minicolumns and recircularizing byligation. The ligation mixtures were transformed into ElectrocompetentSURE cells (Stratagene) and screened for insert size. HA was quantitatedusing the Pharmacia HA Test Kit (DeAngelis, et al., 1993a). UDP-Glcdehydrogenase activity was measured as described (Dougherty and van deRijn, 1993) except cells (from 4.5 ml overnight cultures in dialyzedTHY, washed and resuspended in 0.4 ml buffer) were disrupted byvortexing with an equal volume of washed glass beads (75-150 μm, 5×30 sat 4° C. with 30 s on ice between mixing). The extracts were assayed at30° C. for a UDP-Glc-dependent increase in A₃₄₀ corresponding to NADHproduction. Protein was measured by the Bradford assay (Bradford, 1976)with a BSA standard.

Results

Minicells containing pPDΔPstI produced two nonvector-derived proteins,the intact 42-kDa protein and a 29-kDa truncated version of HasB (FIG.8). The deleted hasB gene product is predicted to be 23-kDa based on thesequence. When transformed into SURE or X1448 cells, pPDi˜PstI could notdirect HA synthesis (Table III). On the other hand, E. coli K5transformed with pPDΔPstI could produce HA (Table III). This observationshould be the result of the endogenous UDP-Glc dehydrogenase, which isresponsible for producing UDP-GlcA needed for K5 capsular polysaccharidesynthesis, substituting for the nonfunctional streptococcal enzyme. Toverify this, the present inventors assayed strains with the variousplasmid constructs for UDP-Glc dehydrogenase activity (Table III).Indeed, all K5 cultures, including those with vector pAT19 alone,demonstrated this activity. SURE or X1448 cells with plasmids encodingan intact 4S-kDa protein possessed elevated enzyme activity, whereascells with the pPDΔPstI plasmid possessed levels similar to host cellsalone.

TABLE III HA Production and UDP-Glc Dehydrogenase Activity in E. coliStrains Containing Various pPD41 Constructs HA UDP-Glc DH PROTEIN^(a)STRAIN PLASMID ng/μl^(b) pmol/min/μg^(c) HasA HasB SURE 0 3 − −PD4I_(Δ)S 61 19 + + PD4I_(Δ)7 0 7 − + PD41_(Δ)PStI 0.2 0.8 + −PD41_(Δ)EcoRV 0 14 − + X1448 AT19 0 0.8 − − PD4I~5 21 17 + + PD4I~7 0 6− + PD4I~PstI 0.6 2.5 + − PD4I~EcoRV 0 10 − + KS (Bi8337-41) AT19 0 12 −− PD4I~S 253 12 + + PD41~7 0 17 − + PD4I~PstI 49 13 + − ^(a)presence orabsence of intact HasA (42 kDa) or HasB (45 kDa) protein as predicted bysequence data and observed by minicell analysis. ^(b)spent culturemedium and SDS cell extracts were pooled and assayed; values arenomalized to 1 A₆₀₀ unit of cells/ml. ^(c)NADH production was measuredin soluble cell extracts in the presence of UDP-G1c. There was noactivity in the absence of UDP-G1c.

These above results demonstrate that the hasA gene product, HasA, is the42-kDa protein, and the HA synthase. The 45-kDa protein derived fromhasB, is the UDP-Glc dehydrogenase (Dougherty and van de Rijn, 1993).Furthermore, studies confirm that the 42-kDa protein has both UDP-GlcNAcand UDP-GlCA glycosyl

transferase activities. Crude membranes from the various constructs showHA synthase activity only in cells with the intact hasA gene.UDP-¹⁴C-GlcA or UDP-³H-GlcNAc are incorporated intohyaluronidase-sensitive product only in the presence of UDPGlcNAc orUDP-GlCA, respectively. This incorporation is decreased by >⁹⁸% ifUDP-GalNAc or UDP-Glc are substituted for UDP-GlcMAc or if UDP-Glc orUDP-GalA are substituted for UDPGlcA.

Dougherty and van de Rijn (Dougherty and van de Rijn, 1993) proposed intheir later model that three ORFs (hasA, hasb, and hasC) are involved inHA biosynthesis. The inventors found that the S43 strain HasB is 99.8%identical at the nucleotide level to their GAS strain HasB sequence;there was perfect conservation at the protein level (not shown). Theregion containing the hasA and hasH genes (Dougherty and van de Rijn,1993) possesses a restriction map consistent with the two ORFs found inpPD41ΔS (FIG. 6). The inventors also found that a putative HasC gene ispresent in S43, but is not required for HA biosynthesis. Neither theHasB nor HasC proteins are needed when both sugar nucleotide precursorsare present.

HA synthase possesses/significant homology with the nodC gene product ofRhizobiumi (DeAngelis, et al., 1994). NodC is a membrane enzyme thatsynthesizes chitin-like (poly-B-1,4-GlcNAc backbone) oligomers (Lerouge,et al., 1990) which is a very analogous activity to that ofstreptococcal HA synthase. NodC possesses several stretches of residuesthat are identical or similar to the HA synthase. Overall the twoproteins are 30.6% identical. The hydropathy plots of the two proteinsare very comparable, including three predicted transmembrane segments inthe same location near the carboxyl terminus (DeAngelis, et al., 1994).Other proteins with homology to HA synthase include DG42 from Xanopuslaavis, yeast chitin synthase II, and an associated protein CSH2(Bulawa, 1992; DeAngelis, et al., 1994). The 52-kDa protein described byPrehm and coworkers (Prehm and Mausolf, 1986; Lansing, et al., 1993) isnot homologous to HasA or these other proteins. The gene cloned by theseworkers is not the HA synthase gene.

hasA and hasH are the only exogenous genes required to direct HAbiosynthesis in most bacteria, due to the presence of one of the sugarnucleotide precursors of HA, UDP-GlcNAc, which is necessary for cellwall formation. In cells that make both UDP-GlcNAc and UDP-G1cA only HAsynthase, the gene product of hasA, HasA, is needed to polymerize the HApolysaccharide (DeAngelis, et al., 1993b)

Example V Large Scale Production of Hyaluronic Acid

This example is directed to the engineering of a bacterium thatoverproduces and secretes large quantities of HA, which can then bepurified from the medium. An engineered organism that overproduces HAwill make it cheaper to produce larger quantities of HA than presentlypossible. Reduced HA production costs will increase the number and typeof commercially viable products that can be developed.

The present inventors have cloned and sequenced a 3,200 base pairStraptococca DNA fragment that confers on recipient bacteria the abilityto make HA as described in Examples II, IV and V. Analysis of this locusrevealed the presence of two tandem genes (FIG. 6). Transformation ofmutant, capsule-deficient Streptococcus cells with these two genesrestored their ability to make HA. Most importantly, putting these twogenes into very different bacteria such as Escherichia coli orEnterococcus faecalis also allowed these bacteria to produce HA. Thiswas shown visually by noting the presence of a new capsule of HAsurrounding the cells (FIG. 2), and biochemically by using a specificassay to detect and quantitate HA (Tables II and III) This resultindicates that after it receives the hasA and hasH genes, any type ofbacteria will be able to make HA.

The nucleotide sequence of this 3.2 kb Straptococcal DNA fragment showedthat the hasB gene encodes the enzyme UDP-glucose dehydrogenase, whichis required for the cell to make UDPglucuronic acid (UDP-G1cA), one ofthe two sugar precursors needed for HA biosynthesis. The second sugarprecursor needed for HA synthesis is UDP-N-acetylglucosamine(UDP-GlcNAc), present in all bacteria and required for cell wallsynthesis. The dehydrogenase gene was also reported by others (Doughertyand van de Rijn, 1993).

The inventors have found that any cell containing a functional UDP-Glcdehydrogenase and a functional HA synthase can make HA. Not all E. colistrains normally have the dehydrogenase. Those that do not will not havethe UDP-GlcA needed for HA synthesis, whereas those that have thedehydrogenase (such as the K5 strain) have both sugar precursors neededfor HA synthesis. When only the functional synthase was present, the K5cells made HA, but the other strains did not (Table III). In no case didrecipient bacteria make HA without a functional HA synthase gene, HasA.

To make a bacterial strain overproduce HA one may place one or morecopies of both the HA synthase gene (and dehydrogenase gene if desired)into an appropriate recipient cell with functional promoters, ribosomebinding sites etc. One would then find the best bacterial recipient,gene copy number and mode of gene regulation.

At each stage of development one can construct one or several bacterialstrains containing the cloned HA synthase gene under the control ofdifferent regulatory elements for expression of the gene. Constructswill also be made containing combinations of the dehydrogenase andsynthase genes in various copy numbers. These bacterial strains will betested in small scale fermentation trials. One would then increase theproduction scale by studying fermentation cultures first on a“bench-scale” (2 liters), then “pilot” (200 liters) and finally a“commercial” scale (15,000 liters)

The strains will be assessed for their growth characteristics and theirability to produce HA. The amount, size and stability of the HA will bedetermined by standard testing procedures known to those of skill in theart. There is significant interest in making the highest MW HA possible,since many biomedical applications for HA require the polymer to be verylong (high MW). It is likely that separate strains can be constructed toachieve production of HA of different average sizes.

Using Bacillus subtilis as a host cell offers distinct advantages forbiotechnology and HA production. For example, the lack of endotoxinproduction by these cells is a big advantage in terms of FDA approvaland the ease of purifying the final product (vs Streptococcus). Thegenetics of B. subtilis has also been extensively studied. Furthermore,these cells make both sugar precursors needed for HA synthesis (Iwasaki,et al., 1989). The inventors have observed that the plasmid pPD41Δ5directs production of HA in B. subtilis strain 1A1 (with a productionrate of at least 500 mg/l of a culture having an OD₆₀₀ of 1).

This recombinant construct can be grown in a very simple and inexpensivegrowth media, such as Spizizen's media (2 gr. (NH₄)₂SO₄, 14 gr. K₂HPO₄,6 gr. KH₂PO₄, 1 gr. Sodium Citrate, and 5 gr. of glucose, per liter ofwater (Ausbel, et al., 1987)) supplemented with tryptophan (0.1 gr/ltr)and erythromycin (8 mg/ltr). On the other hand, Streptococcus bacteriamust be grown on a more complex media that is either expensive and/orcontains large molecules that contaminate HA preparations from spentcultures. These results indicate that B. subtilis is a preferred hostfor the overproduction of HA. One can engineer a B. subtilis strain thatproduces a larger amount of HA than is produced by streptococcalstrains, because the latter may possess low levels of hyaluronidase,which degrade HA.

Therefore, initial efforts are to introduce the HA synthase gene into anasporogenic strain of B. subtilis on a compatible plasmid, and also byfacilitated integration using methods described by others (Smith andYoungman, 1992; Prozorov, et al., 1987; Lewandoski and Smith, 1988;Ausubel, et al., 1987). One would then isolate cells containing variouscopies (say 1, 3 and 5) of the synthase gene and verify that they makeHA. The use of alternate promoters derived from B. subtilis can also bedetermined.

Based on the results of the production studies one can then modify orbegin subsequent rounds of bacterial constructs. For example, one maydecide to assess the effect of having different numbers of the synthasegene and only one-copy of the dehydrogenase gene. Thus, one can“fine-tune” the desired bacterial construct by successive testing andredesigning in order to optimize the quantity and quality of HAproduced.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Ausubel, F. M., et al. 1987. Current Prot. in Mol. Biol. Greene &    Wiley-Intersci., NY.-   Barson (1986), J. Pediatr. Orthop., 6:346-348.-   Benjamin at al. (1976), J. Pediatr., 89:254:256.-   Bitter, T. and Muir, H. (1962) Anal. Biochem. 4:330-334.-   Bulawa, C. E. (1992) Mol. Cell. Biol. 12:1764-1776.-   Caparon, M. G. and Scott, J. R. (1991) Meth. Enzymol. 204:556-586.-   Clewell, D. B., Flannagan, S. E., Ike, Y., Jones, J. M., and    GawronBurke, C., (1988) J. Bacteriol. 170:3046-3052.-   Collins, C. H. and Lyne, P. M., (1976) Microbiological Methods,    Butterworths, Boston, Mass., p. 110.-   Dao et al. (1985), Appl. Envir. Microbiol., 49:115-119.-   DeAngelis, P. L., Papaconstantinou J., and Weigel, P. H. (1993a) J.    Biol. Chem., 268:20, 14568-14571.-   DeAngelis, P. L., Papaconstantinou J., and Weigel, P. H. (1993b) J.    Biol. Chem., 268:26, 19181-19184.-   DeAngelis, P. L., Yang, N., and Weigel, P. H. (1994) Biochemical and    Biophysical Research Communications, Vol. 199, No. 1, pp. 1-20 (Feb.    28, 1994).-   Dinn (1971), J. Ir. Med, Assoc., 64:50-51.-   Dochez, A. R., Avery, O. T., and Lancefield, R. C. (1919) 3. Exp.    Med. 30:179-213.-   Dougherty, B. A. and van de Rijn, I. (1992) J. Exp. Med.    175:1291-1299.-   Dougherty, B. A. and van de Rijn, I. (1993) J. Biol. Chem.    268:7118-7124.-   Dunny, G. M., Lee, L. N., and LeBlanc, D. J. (1991) Appl. Environ.    Microbiol. 57: 1194-1201.-   DuPont Biotech. Update, 4, #4, July 1989.-   Ehtesham, N. Z. and Hasnain, S. E. (1991) BioTechniques 11:718-721.-   European Patent Application EP144019.-   European Patent Application EP266578.-   European Patent Application EP244757.-   Evered, D. and Whelan, J. (eds.) 1989. The Biology of Hyaluronan.    Wiley, Chichester,-   U. K. Fiers at al., Mature, 273:113 (1978).-   Gawron-Burke, C. and Clewell, D. B. (1984) J. Bacteriol.    159:214-221.-   Gren, E. J. (1984) Biochimie 66:1-29.-   Guerrero, R., Pedros-Alio, C., Schmidt, T. M., and Mas, J. (1985),    Microbiologia, 1:53-65.-   Gupta, D. S. Jann, B., Schmidt, G., Golecki, J. R., Orskov, I.,    Orskov, F., and Jann, K., (1982) FEMS Microbiol. Letters 14:75-78.-   H{dot over (a)}kansson S., and Holm S. E., (1986) Acta Path.    Microbiol. Immunol. Scand. Sect. B 94:139-43.-   Hirsch at al. (1960), J. Exp. Med., 111:309-322.-   Ishimoto et al. (1967), Biochim. Biophys. Acta, 148:296-297.-   Iwasaki, H., Araki, Y., Kaya, S, and Ito, E. (1989) Eur. J. Biochem.    178:635-641.-   Jones, Genetics, 85:12 (1977).-   Kass at al. (1944), J. Exp. Med., 79:319-330.-   Kendall, F., Heidelberger, M., and Dawson, M. (1937) J. Biol. Chem.    118:61-69.-   Kyte & Doolittle (1982) J. Mol. Biol. 157:105-132.-   Laemmli, U. K. (1970) Nature 227:680-68S.-   Lansing, M., Lellig, S., Mausolf, A., Martini, I., Crescenzi, F.,    O'Regan, M., and Prehm, P. (1993) Biochem. J. 289:179-184.-   Laurent, T. C. and Fraser, J. R. E. (1992) FASE3J. 6:2397-2404.-   Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G.,    Prome, J. C., and Denarie, J. (1990) Nature 344:781-784.-   Lewandoski, M. and Smith, I. (1988) Plasmid 20:148-154.-   MacLennan, A. P. 1956, J. Gen. Microbiol. 14:134-142.-   Markovitz, A., Cifonelli, J. A., and Dorfman, A. (1959) J. Biol.    Chem. 234:2343-2350.-   Markovitz et al. (1962), J. Biol. Chem., 237:273-279.-   Matsumura, P., Silverman, M. and Simon, M. (1977) J. Bacteriol. 132:    996-1002.-   Maxam et al. (1980), Meth. Enzymol., 65:499.-   Meagher, R. B., Tait, R. C., Betlach, M. and Boyar, H. W. (1977)    Cell 10: 521-536.-   Messing et al. (1981), Nucl. Acids Res., 9:309.-   Min, H. and Cowman, M. K. (1986) Anal. Biochem. 155:275-285.-   Ng, K. F. and Schwartz, N, B. (1989) J. Biol. Chem. 264:11776-11783.-   O'Connor, S. P. and Cleary, P. P. (1987) J. Infect. Dis. 156:    495-504.-   Prehm, P. (1983) Biochem. J. 211:181-189.-   Prehm, P. and Mausolf, A. 1986. Biochem. J. 235:887-889.-   Prozorov, A. A., Poluektova, E. U., Savchenko, G. V.,    Nezmetdinova, V. Z. and Khasanov, F. K. (1987) Gene 57:221-227.-   Quinn, A. W. and Singh, K. P., (1957) Biochem. J. 95:290-201.-   Rizkallah et al. (1988), J. Infect. Dis., 158:1092-1094.-   Rotta (1988), APMIS Suppl., 3:3-7.-   Sambrook, J., Fritsch, E. F., and Maniatis, T., (1989) Molecular    Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor    Laboratory, Cold Spring Harbor, N.Y.-   Sangar at al. (1977), Proc. Natl. Acad. Sci. USA, 74:5463-5467.-   Schaechter, M., Medoff, G., and Schlessingar, D., editors, (1989)    Mechanisms of Microbial Disease, Williams and Wilkins, Baltimore,    Md.-   Scott et al. (1964), Histochemie, 4:73-85.-   Siefkin et al. (1983), J. Clin. Microbiol., 17:386-388.-   Smith, K. and Youngman, P. (1992) Biochimia 74:705-711.-   Stoolmiller et al. (1969), J. Biol. Chem., 244:236-246.-   Studier et al. (1990) Meth. Enzymol. 185:60-89.-   Sugahara et al. (1979), J. Biol. Chem., 254:6252-6261.-   Tengblad, A. (1980) Biochem. J. 185:101-105.-   Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)-   Trieu-Cuot, P., Carliar, C., Poyart-Salmeron, C. and    Courvalin, P. (1991) Gene 102:99-104.-   Triscott, M. X. and van de Rijn, I. (1986) J. Biol. Chem.    261:6004-6009.-   van de Rijn, I. and Drake, R. R. (1992) J. Biol. Chem. 267:    24302-24306.-   Wessels, M. R., Moses, A. E., Goldberg, J. B., and DiCesare, T.    J., (1991) Proc. Natl. Acad. Sci. USA, 88:8317-8321.-   Whitnack et al. (1981), Infect. Immun., 31:985-991.

1. A purified nucleic acid segment encoding hyaluronan (HA) synthase. 2.A recombinant vector comprising the purified nucleic acid segment ofclaim
 1. 3. A recombinant host cell comprising the recombinant vector ofclaim
 2. 4. A method of producing HA, comprising the steps of: providingthe recombinant host cell of claim 3; growing the recombinant host cellin a medium to secrete hyaluronic acid; and recovering the secretedhyaluronic acid.